Isbn 9789514290053

OULU 2009
D 1002
D 1002
U N I V E R S I T Y O F O U L U P. O. B . 7 5 0 0 F I - 9 0 0 1 4 U N I V E R S I T Y O F O U L U F I N L A N D
ACTA U N I V E R S I T AT I S O U L U E N S I S
ACTA
A C TA U N I V E R S I TAT I S O U L U E N S I S
S E R I E S E D I T O R S
Virve Pkknen
D MEDICA
ASCIENTIAE RERUM NATURALIUM

EXPRESSION PROFILING
Virve Pkknen
Professor Mikko Siponen
BHUMANIORA
University Lecturer Elise Krkkinen
OF HUMAN PULP TISSUE
CTECHNICA
AND ODONTOBLASTS
Professor Hannu Heusala IN VIVO AND IN VITRO
DMEDICA
Professor Olli Vuolteenaho
ESCIENTIAE RERUM SOCIALIUM

Senior Researcher Eila Estola
FSCRIPTA ACADEMICA
Information officer Tiina Pistokoski
GOECONOMICA
University Lecturer Seppo Eriksson
EDITOR IN CHIEF
PUBLICATIONS EDITOR
Publications Editor Kirsti Nurkkala FACULTY OF MEDICINE,
INSTITUTE OF DENTISTRY,
UNIVERSITY OF OULU;
ISBN 978-951-42-9004-6 (Paperback) INSTITUTE OF DENTISTRY,
ISBN 978-951-42-9005-3 (PDF) UNIVERSITY OF HELSINKI
ISSN 0355-3221 (Print)
ISSN 1796-2234 (Online)
ACTA UNIVERSITATIS OULUENSIS
D Medica 1002
VIRVE PKKNEN
EXPRESSION PROFILING OF HUMAN

PULP TISSUE AND ODONTOBLASTS
IN VIVO AND IN VITRO
Academic dissertation to be presented, with the assent of

the Faculty of Medicine of the University of Oulu, for
public defence in Auditorium 1 of the Institute of
Dentistry (Aapistie 3), on January 30th, 2009, at 12 noon
O U L U N Y L I O P I S TO, O U L U 2 0 0 9
Copyright 2009
Acta Univ. Oul. D 1002, 2009
Supervised by
Professor Tuula Salo
Professor Leo Tjderhane
Reviewed by
Professor Matti Nrhi
Docent Pirkko Pussinen
ISBN 978-951-42-9004-6 (Paperback)

ISBN 978-951-42-9005-3 (PDF)
http://herkules.oulu.fi/isbn9789514290053/
ISSN 0355-3221 (Printed)
http://herkules.oulu.fi/issn03553221/
Cover design
Raimo Ahonen
OULU UNIVERSITY PRESS

OULU 2009
Pkknen, Virve, Expression profiling of human pulp tissue and odontoblasts in
vivo and in vitro
Faculty of Medicine, Institute of Dentistry, University of Oulu, P.O.Box 5281, FI-90014
University of Oulu, Finland; Institute of Dentistry, University of Helsinki, P.O.Box 41, FI-
00014 University of Helsinki, Finland
Acta Univ. Oul. D 1002, 2009
Oulu, Finland
Abstract
Dentin forms the hard tissue portion of the dentin-pulp complex, while the dental pulp is soft
connective tissue that retains the vitality of the dentin. Odontoblasts form the outermost cell layer
of pulp and play a central role during dentin formation by producing and mineralizing the dentin
matrix. The understanding of the defensive reactions in the dentin-pulp complex is limited.
Information about the transcriptome and proteome of pulp tissue and odontoblasts would facilitate
understanding of their functions during health and disease. The aim of this study was to investigate
the expression profiles of human pulp tissue and odontoblasts in vivo and in vitro using large-scale
expression analysis methods. Also, the suitability of these methods in pulp biological research in
vivo and in vitro was evaluated.
cDNA microarray revealed only minor variation and 2-D electrophoresis combined with mass
spectrometry revealed no differences between healthy and carious teeth pulp tissue in vivo. The
effect of transforming growth factor 1 (TGF-1) on pulp and odontoblasts was studied in vitro
using oligonucleotide-based microarrays, and marked changes in the transcriptome were revealed,
especially in the expression of chemokine- and cytokine-related genes. Transiently increased
interleukin expression was confirmed at the protein level by antibody array.
DNA microarray analysis of native pulp tissue and odontoblasts was used to search for
potential odontoblast markers. Only one gene related to extracellular matrix organization and
biogenesis, matrilin 4, and two expressed sequence tags (ESTs), which represent transcribed
sequences encoding possibly unknown genes, were identified in odontoblasts but not in pulp.
Analysis of mature native odontoblasts and cultured odontoblast-like cells by DNA microarray
revealed a high similarity (84%) between native and cultured cells. Also, differential expression
levels of selected neuronal proteins were observed and confirmed at the mRNA and protein levels.
In conclusion, microarray is a powerful tool for pulp biology, especially for in vitro studies.
TGF-1 was revealed as a potent regulator of proinflammatory responses in the dentinpulp
complex. In addition, several potential odontoblast markers were identified by microarray, and the
similarity of cultured odontoblast-like cells used in the study with native odontoblasts was
confirmed.
Keywords: dental pulp, gene expression profiling, humans, odontoblasts, TGF-

Acknowledgements
This study was carried out at the Institute of Dentistry at the University of Oulu. I
want to express my gratitude for my supervisors, Professor Leo Tjderhane and
Professor Vice-dean Tuula Salo, for their guidance and for all the things I have
learned from them.
I wish to acknowledge the official referees of this thesis, Professor Matti
Nrhi and Docent Pirkko Pussinen, for their careful review of the manuscript and
their helpful comments. I also thank Ms. Sandra Hnninen for the careful
language revision of the manuscript.
I want to thank all my co-authors. I am especially thankful to Dr. Jussi
Vuoristo for his invaluable help with microarray and good advice. Professor
Henry Magloire and Professor Franoise Bleicher in Lyon are acknowledged for
fruitful collaboration. Im also grateful for the fun and edifying two weeks I spent
in the Laboratoire Dveloppement et Rgnration des Tissus Dentaires.
I wish to thank Dr. Terttu Sippola, Dr. Juha Alatalo and Dr. Ari Lnsineva and
their teams for providing the teeth, which were absolutely necessary for this work.
Thanks go to all current and former members of the MMP group: Ibrahim
Bello, Jarkko Korpi, Sini Nurmenniemi, Pia Nyberg, Heidi Palosaari, Mataleena
Parikka, Emma Piril, Aleksi Rytknen, Merja Sulkala, Meeri Sutinen, Anu
Vnnen, Merja Ylipalosaari and Pirjo strm for creating friendly working
atmosphere. I am very grateful to Maija-Leena Lehtonen and Sanna Juntunen for their
skilful technical assistance as well as to all the staff at the Institute of Dentistry who
have helped me with various practical matters and occasionally answered my weird
questions.
I warmly thank my sister Mirva for her help with the figures of this thesis,
and my parents and Artsi for support. I also want to thank my friends, and in
particular the ones who formed the (increasingly) critical mass for the coffee and
lunch breaks.
This study was supported by Academy of Finland, Oulu University Hospital,
Finnish Cultural Foundation, Oulu University Pharmacy Foundation, Orion
Farmos Research Foundation, Finnish Dental Society Apollonia, and COST
action b23.
Oulu, December 2008 Virve Pkknen
5
6
Abbreviations
BIA/MS biomolecular interaction analysis/mass spectrometry
BMP bone morphogenetic protein
bp base pair
CCL chemokine, CC motif, ligand
CGRP calcitonin gene-related peptide
COL collagen
cDNA complimentary DNA
cRNA complementary RNA
CSF colony-stimulating factor
CXCL chemokine, CXC motif, ligand
DARC duffy blood group antigen
DC dendritic cell
DLK delta-like protein
DPP dentin phosphoprotein
DSPP dentin sialophosphoprotein
DSP dentin sialoprotein
ECM extracellular matrix
EDG4 G protein coupled receptor
EST expressed sequence tag
ET-2 endothelin 2
GAL galanin
GDF-1 embryonic growth/differentiation factor 1
GEO Gene Expression Ontology
GO gene ontology
IEF isoelectric focusing
IFN interferon
IGF insulin-like growth factor
IL interleukin
JAG jagged homolog
LTA lipoteichoic acid
LPS lipopolysaccharides
MALDI-TOF matrix-assisted laser-desorption/ionization time-of-flight
MATN4 matrilin 4
MIAME minimum information about microarray experiments
MMP-13 matrix metalloproteinase 13
7
MPSS parallel signature sequencing
mRNA messenger RNA
MSP macrophage-stimulating protein
MS mass spectrometry
NDP nucleoside diphosphate
NF 90 nuclear factor 90
NGF-2 nerve growth factor 2
NGR neurogranin
NK neurokinin
NK cell natural killer cell
NT neurotensin
OSM oncostatin M
PAX3 paired box protein 3
PBS phosphate-buffered saline
PBST phosphate-buffered saline buffer containing Tween 20
PLA2 phospholipase A2
PLX B3 Plexin related protein B3
PlGF placental growth factor
PNOC pronociceptin
RT-PCR reverse transcriptase polymerase chain reaction
SAGE serial analysis of gene expression
SDS sodium dodecyl sulfate
PAGE polyacryleamide gel electrophoresis
qPCR quantitative PCR
SFN stratifin
SMAD mothers against decapentaplegic, drosophila, homolog
SPR surface plasmon resonance
SSH suppression subtractive hybridization
SSTR1 somatostatin receptor 1
TDGF-1 teratoma-derived growth factor
TEF-SII transcription elongation factor SII
TIE-2 TEK tyrosine kinase
TGF transforming growth factor
TNF tumor necrosis factor
TNFR-1 tumor necrosis factor receptor-1
TLR toll-like receptor
UCE-E2 ubiquitin-conjugating enzyme-E2
8
VEGF vascular endothelial growth factor
2-D two-dimensional
alpha
beta
gamma
9
10
List of original publications
The thesis is based on following articles, which are referred to in the text by
Roman numerals:
I Pkknen V, Ohlmeier S, Bergmann U, Larmas M, Salo T & Tjderhane L (2005)
Analysis of gene and protein expression in healthy and carious tooth pulp with cDNA
microarray and two-dimensional gel electrophoresis. Eur J Oral Sci 113: 369379.
II Pkknen V, Vuoristo JT, Salo T & Tjderhane L (2008) Comparative gene
expression profile analysis between native human odontoblasts and pulp tissue. Int
Endod J 41:117127.
III Pkknen V, Vuoristo J, Salo T & Tjderhane L (2007) Effects of TGF-beta1 on
interleukin profile of human dental pulp and odontoblasts. Cytokine 40: 4451.
IV Pkknen V, Bleicher F, Carrouel F, Vuoristo JT, Salo T, Wappler I, Couble ML,
Magloire H, Peters H & Tjderhane L (2009) General expression profiles of human
native odontoblasts and pulp-derived cultured odontoblast-like cells are similar but
reveal differential neuropeptide expression levels. Arch Oral Biol 54: 5562.
11
12
Contents
Acknowledgements 5
Abbreviations 7
List of original publications 11
Contents 13
1 Introduction 17
2 Review of the literature 19
2.1 Structure of the tooth............................................................................... 19
2.2 Dentin-pulp complex............................................................................... 19
2.2.1 Pulp tissue..................................................................................... 20
2.2.2 Odontoblasts................................................................................. 21
2.2.3 Dentin ........................................................................................... 22
2.2.4 Inflammatory response of the dentin-pulp complex..................... 23
2.3 Cytokines in the dentin-pulp complex .................................................... 24
2.3.1 Inflammatory cytokines in the dentin-pulp complex.................... 25
2.3.2 Cytokine response of the dentin-pulp complex to caries.............. 28
2.4 Neuropeptides in the dentin-pulp complex ............................................. 31
2.5 Transcriptome and proteome of the dentin-pulp complex....................... 31
2.5.1 Transcriptomics ............................................................................ 32
2.5.2 Proteomics .................................................................................... 36
2.5.3 Transcriptomics and proteomics in pulp biology ......................... 37
3 Aims of the study 39
4 Materials and methods 41
4.1 Samples ................................................................................................... 41
4.1.1 Collection of mature native teeth and native odontoblast
and pulp samples (IIV) ............................................................... 41
4.1.2 Pulp tissue and odontoblast cultures (II, III) ................................ 42
4.1.3 Cultured odontoblast-like cells (IV) ............................................. 42
4.1.4 Mouse bone samples (II) .............................................................. 43
4.2 Extraction of RNA and proteins (IIV)................................................... 43
4.3 Large-scale analysis of gene expression ................................................. 43
4.3.1 cDNA microarray (I) .................................................................... 43
4.3.2 GeneChip microarray (II, III, IV)................................................. 44
4.4 RT-PCR (I, II, III).................................................................................... 46
4.5 Real-time quantitative PCR (IV)............................................................. 47
4.6 Proteomics............................................................................................... 47
13
4.6.1 2-D electrophoresis and mass spectrometry (MS) (I) ................... 47
4.6.2 Antibody array (III) ...................................................................... 48
4.7 Western blot (II) ...................................................................................... 49
4.8 Immunofluorescence (IV) ....................................................................... 49
5 Results 51
5.1 The effects of caries on the expression profile of pulp tissue (I)............. 51
5.1.1 The effects of caries on the mRNA profile of pulp tissue............. 51
5.1.2 The effects of caries on the protein profile ................................... 52
5.2 Repeatability of Affymetrix GeneChip arrays (II) .................................. 53
5.3 The effects of TGF-1 on the general expression profile of pulp
tissue and odontoblasts (III) .................................................................... 53
5.4 Interleukin profile of pulp tissue and odontoblasts (I, III) ...................... 54
5.4.1 Interleukin profile of native pulp tissue........................................ 54
5.4.2 Effects of TGF-1 on the interleukin expression profile of
pulp tissue (III) ............................................................................. 54
5.4.3 Interleukin profile of native odontoblasts (III) ............................. 55
5.4.4 Effect of TGF-1 on the interleukin expression profile of
odontoblasts (III) .......................................................................... 56
5.5 Differences between the general expression profiles of native
pulp tissue and odontoblasts (II) ............................................................. 56
5.6 Potential odontoblast markers (II, III)..................................................... 57
5.6.1 Matrilin 4 and collagen type XI 1............................................... 57
5.6.2 Expressed sequence tags (EST) .................................................... 57
5.6.3 Dentin sialophosphoprotein (DSPP)............................................. 57
5.7 Comparison of the general expression profiles of mature native
odontoblasts and newly differentiated cultured odontoblast-like
cells (IV) ................................................................................................. 58
5.7.1 Expression of selected neuronal proteins in mature and
newly differentiated odontoblasts................................................. 59
6 Discussion 61
6.1 Suitability of large-scale screening methods in pulp-biological
research ................................................................................................... 62
6.1.1 In vivo analysis of the transcriptome and proteome ..................... 62
6.1.2 In vitro analysis of the transcriptome and proteome..................... 64
6.2 Comparison of microarray with other large-scale transcription
screening methods used in pulp biology ................................................. 65
6.3 The effect of caries on the expression profile of pulp tissue ................... 65
14
6.3.1 The effect of caries on the transcriptome of pulp tissue ............... 66
6.3.2 The effect of caries on the proteome of pulp tissue...................... 68
tissue and odontoblasts............................................................................ 68
6.5 Cytokine profile of pulp tissue and odontoblasts .................................... 70
6.5.1 Interleukin profile of pulp tissue .................................................. 70
6.5.2 Interleukin profile of odontoblasts ............................................... 72
6.6 Potential novel odontoblast markers ....................................................... 73
6.6.1 Matrilin 4 and collagen XI 1 ...................................................... 73
6.6.2 The expressed sequence tags detected only in odontoblasts......... 74
6.7 Similarity of cultured odontoblast-like cells to mature native
odontoblasts ............................................................................................ 75
6.7.1 Neuronal protein expression in mature and newly
differentiated odontoblasts ........................................................... 76
Conclusions 79
References 81
List of original publications 95
15
16
1 Introduction
The dentin-pulp complex consists of pulp tissue, which is soft connective tissue
containing nerves and blood vessels, and mineralized hard tissue, dentin. The pulp
maintains the dentin vitality, while odontoblasts, which form the outermost cell
layer of pulp tissue, produce and mineralize dentin. (Ten Cate 1994). Mature
odontoblasts are terminally differentiated cells and unable to proliferate, but pulp
tissue is thought to contain cells capable of differentiating into replacement
odontoblasts when the original odontoblasts have died (Smith 2003, Mitsiadis &
Rahiotis 2004). The encasement of the pulpal cells within dentin causes two
problems: the cells are not accessible for immunohistological study without
demineralization, which may affect the proteins to be studied; and secondly, pulp
is in a low-compliance environment, in which all of the normal inflammatory
reactions cannot take place (Fouad 2002). Thus, the understanding of the
defensive reactions in the dentin-pulp complex is still limited.
Traditional methods used for analysis of mRNA or protein expression of pulp
tissue have the disadvantage of focusing only on few different molecules. Recent
advances in molecular genetics and computational biology have led to rapid
development of various high-throughput methods for analysis of the
transcriptome and proteome, the mRNA and protein profiles of cells and tissues.
However, the use of these methods in pulp biology has been limited. The
transcriptome and proteome of dentin-pulp complex cells reflect their functions,
and thus detailed knowledge of the expression profiles during health and disease
would enable the search for target molecules for treatment and diagnosis.
The aim of the present study was to evaluate the usefulness of the high-
throughput methods for pulp biological research and to analyze the transcriptome
and proteome of human pulp tissue and odontoblasts in vivo and in vitro.
Specifically, the expression profiles of healthy and carious teeth pulp tissues were
compared in vivo. Also, the effect of transforming growth factor 1 (TGF-1) on
pulp tissue and odontoblasts in vitro was investigated. The difference between
mature native odontoblasts and pulp tissue as well as cultured newly
differentiated odontoblasts was analyzed.
17
18
2 Review of the literature
2.1 Structure of the tooth
Anatomically, the tooth can be divided into a crown and a root (Fig. 1). The
crown is covered by enamel, which is acellular and consists of 95 volume
percents of hydroxyapathite crystals, making it the hardest tissue in human body.
The root is covered by cementum, which is 50% hydroxyapathite and has
collagen matrix. Dentin, which forms the bulk of the tooth, is covered by enamel
and cementum. The dentin is 70% mineralized with hydroxyapathite crystals and
its organic matrix is comprised mostly of fibrous collagen. Dental pulp, the soft
tissue maintaining the vitality of the tooth, is encased in dentin. (Ten Cate 1994,
Okiji 2002).
Fig. 1. Tooth structure modified from Netter 2006.
2.2 Dentin-pulp complex
Together, dentin and pulp form the dentin-pulp complex. Odontoblasts, the cells
that form and mineralize the dentin matrix, are located as the outermost cell layer
at the boundary of pulp tissue and predentin. (Ten Cate 1994, Okiji 2002).
19
2.2.1 Pulp tissue
Pulp is composed of soft connective tissue containing blood vessels and nerves,
and retains the vitality of the dentin-pulp complex (Okiji 2002, Linde & Goldberg
1993). The most abundant cell type in the pulp tissue is fibroblasts, but pulp also
contains endothelial cells, nerve fibers, undifferentiated mesenchymal cells and
various immunocompetent cells (Okiji 2002, Goldberg & Smith 2004).
In coronary pulp, fibroblasts form a cell-rich zone (Hhl cells, sub-
odontoblastic cells) that is separated from the odontoblast layer by a cell-free
zone (zone of Weil). The zone of Weil contains blood capillaries, a rich network
of unmyelinated nerve fibers and fibroblast processes. (Okiji 2002, Linde &
Goldberg 1993). The central connective tissue, known as the pulp proper, which
consists of fibroblasts, larger blood vessels and nerves, is located inwards from
the cell-rich zone (Okiji 2002). Undifferentiated mesenchymal cells, the
progenitor cells of pulp that are thought to be capable of differentiating into
fibroblasts or odontoblasts, are distributed throughout the pulp tissue and
frequently located perivascularly (Okiji 2002, Mjor et al. 2001). In addition,
several types of immunocompetent cells, including dendritic cells, macrophages
and T- and B-lymphocytes, have been detected in pulp (Jontell et al. 1998).
The pulpal extracellular matrix (ECM), which is produced by the fibroblasts,
consists of collagens, proteoglycans and glycoproteins, and its composition is
similar to that in other soft connective tissues (Okiji 2002, Linde 1985). Most of
the collagen in pulp tissue is fibrillar; types I and III collagen comprise 95% of
the total collagen, while non-fibrillar collagens V and VI are found in lesser
amounts (Tsuzaki et al. 1990, Lukinmaa & Waltimo 1992).
Blood vessels and nerves enter the pulp through the apical foramen or
foramina of the root, and then branch coronally (Mjor et al. 2001). The
vasculature is most abundant in the subodontoblastic area, and some capillaries
even enter the odontoblast layer, but not the dentin (Linde & Goldberg 1993,
Kramer 1960, Takahashi et al. 1982). The sensory and sympathetic nerves of pulp
tissue mainly follow the blood vessels as they branch, and form a rich network of
terminal endings in the subodontoblastic region and in the periodontoblastic
spaces of dentinal tubules extending 0.1 mm into dentin in the crown (Byers 1984,
Byers et al. 2003, Mjor et al. 2001). The majority of these nerves are sensory
(Pohto & Antila 1968, Byers & Nrhi 1999, Byers et al. 2003, Haug & Heyeraas
2006). The sensory nerve fibers are especially numerous near the pulp horn tips
20
(Byers et al. 2003). Odontoblasts and the subodontoblastic area are not normally
innervated by symphatic nerves (Uddman et al. 1998).
2.2.2 Odontoblasts
Odontoblasts, the cells responsible for dentin formation, are located along the
periphery of the pulp chamber as a single cell layer. They are elongated,
terminally differentiated, post-mitotic cells. During development, odontoblast
precursors migrate from the neural crest as a part of the ectomesenchymal cell
population that participates in the formation of the oral cavity (Garant 2003b,
Arana-Chavez & Massa 2004). Pre-odontoblasts exit the cell cycle and
differentiate into odontoblasts, due to reciprocal signaling with the adjacent cells
and the extracellular matrix of the dental epithelium. (Garant 2003b, DSouza
2002). Mature functional odontoblasts are polarized cells with the cell body
facing towards the pulp tissue and a single cell process extending into the
predentin and dentin inside a dentinal tubule (Okiji 2002, Linde & Goldberg
1993). Mature odontoblasts that have exited the cell cycle are unable to
proliferate, but pulp tissue is thought to contain cells capable of differentiating
into replacement odontoblasts when the original odontoblasts have died (Smith
2003, Mitsiadis & Rahiotis 2004). A cell line capable of differentiating into
various cell types and generating dentin-like mineralized structures has been
characterized in pulp tissue (Gronthos et al. 2002, Gronthos et al. 2000). When
transplanted ex vivo into mouse, these cells form a layer of aligned odontoblast-
like cells with their processes similarly oriented and extending into the hard tissue
they have generated. However, when cultured in vitro these cells do not have the
morphological characteristics of odontoblasts. The cell line established by Couble
et al. (2000) has more odontoblast-like morphology with the typical cellular
processes of odontoblasts, and the cells align into the same orientation when
grown on plastic in vitro. These cells also form dentin-like mineralized zones
(Couble et al. 2000).
In addition to their function in dentin formation, odontoblasts have been
suggested to participate in other important functions, including the sensory
response of tooth. The findings that mechanosensitive potassium channels (Allard
et al. 2000, Magloire et al. 2003) and voltage-gated calcium channels are present
in the odontoblast plasma membrane (Lundgren & Linde 1997, Davidson & Guo
2000) support this function. However, though adhesion-like contacts between
nerve fibers and odontoblasts have been detected, no synaptic structures have
21
been observed (Byers 1984, Byers & Nrhi 1999). It has also been suggested that
odontoblasts may play a role in the immune response of the tooth. This hypothesis
is supported by findings that odontoblasts consistently produce components of
innate and adaptive immunity (Veerayutthwilai et al. 2007, Dommisch et al.
2005), and odontoblasts can be induced to express cytokines and chemokines
(Veerayutthwilai et al. 2007, Levin et al. 1999).
2.2.3 Dentin
Dentin is a mineralized connective tissue, and its characteristics are largely

determined by its mineralized extracellular matrix. On average, dentin consists of
50% minerals (mainly hydroxyapathite), 30% organic matrix and 20% water.
However, the distribution of these constituents varies in different parts and types
of dentin. (Ten Cate 1994, Linde & Goldberg 1993)
The process of dentin formation is called dentinogenesis. Primary dentin,
deposited during the rapid phase of physiological dentin formation by
odontoblasts, forms the bulk of the tooth (Linde & Goldberg 1993). The dentin
matrix is gradually organized and then mineralized (Garant 2003a), except for a
1020 m wide zone of predentin that remains non-mineralized and contains
collagen and other ECM molecules (Linde & Goldberg 1993). When odontoblasts
produce dentin, they move in a centripetal direction leaving their cell processes in
the dentin. As a consequence, dentinal tubules are formed that also contain
proteins produced by odontoblasts, serum proteins and dentinal fluid (Arana-
Chavez & Massa 2004, Garant 2003a). The true length of odontoblast processes
in mature teeth remains undetermined. The odontoblast processes extend at least
to the middle of the dentin, while some analyses indicate that they may extend to
the dentin-enamel junction (Garant 2003a). It has been suggested that odontoblast
processes may contract towards the odontoblast cell body when they come in
contact with the fixatives used to prepare the samples for electron microscopic
studies (Garant 2003a, La Fleche et al. 1985). The timing of primary
dentinogenesis turning into secondary dentinogenesis has been defined as the time
when the tooth reaches occlusal contact (Linde & Goldberg 1993), the time of the
apex closure (Garant 2003a) or the tooth eruption (Mjor et al. 2001). Secondary
dentin is deposited, at a lower rate than primary dentin, throughout the pulp space
by the original odontoblasts (Garant 2003a, Smith 2002). The term tertiary
dentinogenesis is used to describe the dentin formation due to external stimuli.
Tertiary dentin is formed under the site of injury, and the deposition rate is
22
proportional to the degree of injury (Linde & Goldberg 1993, Mjor et al. 2001).
Tertiary dentin is called reactionary dentin when it is formed by odontoblasts
surviving the injury, and reparative dentin when formed by replacement
odontoblasts, which have differentiated from pulp stem cells and migrated to the
site of injury (Garant 2003a, Smith 2002).
The dentin organic matrix is composed of collagenous and noncollagenous
proteins, proteoglycans, glycoproteins and lipids (Linde & Goldberg 1993,
Goldberg & Smith 2004). Collagens are the most common proteins in the dentin
extracellular matrix (90%), type I collagen being the most abundant (Linde &
Goldberg 1993, Goldberg & Smith 2004). Type III collagen and, in lesser
amounts, types V and VI have also been found in predentin or dentin (Goldberg &
Smith 2004). Many noncollagenous proteins are considered to play important
roles in promoting and controlling the mineralization of collagen fibrils and
crystal growth during dentin formation (Butler et al. 2003). Dentin also contains
multiple growth factors, including TGF-1-3, insulin-like growth factors (IGF) -I
and -II, bone morphogenetic proteins (BMPs) and several angiogenic growth
factors (Finkelman et al. 1990, Roberts-Clark & Smith 2000, Bessho et al. 1991,
Cassidy et al. 1997). These dentin-bound growth factors may be released during
processes leading to dentin dissolution, e.g. caries progression, and have the
potential to stimulate reparative dentinogenesis (Smith 2003).
2.2.4 Inflammatory response of the dentin-pulp complex
Invasion of bacteria or bacteria-derived factors into the pulp is a major etiologic

factor in the inflammation of pulp tissue. This invasion can be initiated by e.g.
caries or tooth fracture. (Trowbridge 2002).
Pulpal inflammation caused by caries begins with a low-grade immune
response to bacteria and bacteria-derived factors, and initially resembles a chronic
inflammatory reaction (Trowbridge 2002). The first signs of inflammation can be
observed before the bacteria penetrate into the dentin (Brannstrom & Lind 1965,
Bjorndal & Mjor 2001). Indeed, there is proof that the defensive and
inflammatory reactions occur in the pulp tissue even during the very early phases
of enamel caries (Bergenholtz 2000). Pulpal nerve fibers react to inflammation by
sprouting terminal branches and by altering their neuropeptide, e.g. calsitonin
gene-related peptide (CGRP), production (Byers & Nrhi 1999, Byers et al. 2003).
The sensory nerve fibers play an important role in promoting the arrival of
immunocompetent cells into pulp tissue (Fristad et al. 1997). The initial
23
infiltrating inflammatory cells contain mostly lymphocytes, macrophages and
plasma cells with a minor amount of neutrophils (Trowbridge 2002). The acute
pulpal inflammation, with the migration of abundant amounts of neutrophils
towards the lesion, starts only when bacteria have invaded the reparative dentin
formed under the lesion (Trowbridge 2002, Langeland 1987). The acute
inflammatory response consists largely of a vascular reaction; mediators released
by various cells (e.g. serotonin, histamine and neuropeptides such as substance P,
CGRP, neurokinin A and somatostatin) lead to alterations in pulp blood flow and
increase the capillary permeability (Fouad 2002). Thus, plasma proteins and
neutrophils gain better access to the inflamed area and can neutralize or
phagocytose the irritant (Trowbridge 2002). Different types of T-lymphocytes
(CD4+ helper, CD8+ cytotoxic), macrophages, neutrophils, dendritic cells, and
plasma cells can be found in inflamed pulp tissue, and their numbers increase
when the disease progresses (Jontell et al. 1998). The dendritic cells and also
CD3+ (memory) T-lymphocytes accumulate under the lesion, while the
accumulation of other immunocompetent cells is minor, suggesting that the
dendritic cells and the memory T cells are critical in triggering immunological
reactions of pulp (Jontell et al. 1998). Severe pulpal inflammation may lead to
increased interstitial tissue pressure, which in turn causes necrosis, since pulp is
enclosed inside rigid mineralized structures and thus is a non-compliance
environment (Trowbridge 2002, Heyeraas & Berggreen 1999).
2.3 Cytokines in the dentin-pulp complex
Cytokines are proteins and polypeptides that are produced by a cell and affect the
growth or differentiation of the same or another cell. The term includes e.g.
interleukins (ILs) and chemokines as well as differentiation and growth factors,
but usually excludes endocrine hormones. Cytokines are characterized by their
pleiotropism and redundancy and have vital roles in e.g. differentiation, cell
division, apoptosis and inflammation. Virtually all cell types are capable of
producing some cytokines in response to diverse stimuli. (Oppenheim &
Feldmann 2003). Many cytokines play roles in immune response and immunity
and can be called inflammatory cytokines. Proinflammatory cytokines are
molecules that induce inflammatory reactions, while anti-inflammatory cytokines
have suppressory function in the inflammatory response (Oppenheim &
Feldmann 2003). Classic proinflammatory cytokines include IL-1, IL-12, tumor
necrosis factor (TNF-), granulocyte-macrophage colony-stimulating factor
24
(GM-CSF) and interferon (IFN-), while e.g. IL-4, IL-10, IL-13 have anti-
inflammatory functions (Oppenheim & Feldmann 2003). Chemokines, a group of
small (approximately 8 to 14 kD) structurally related molecules that regulate the
cell trafficking of leukocytes, are another group of major contributors to
inflammation. The chemokines also play crucial roles in the development,
homeostasis, and function of the immune system, and they have additional effects
on cells of the central nervous system and angiogenesis. Chemokines are divided
into two major subfamilies, CXC and CC, based on the arrangement of the first
two of the four conserved cysteine residues. (Palmqvist et al. 2007).
Cytokines have multiple important functions in the dentin-pulp complex
during health and disease. Inflammatory cytokines both pro- and anti-
inflammatory, e.g. several interleukins and chemokines have critical roles in the
immune defense of dental pulp (Fouad 2002). Growth factors, which can be
released from dentin during caries, play a central role in the regulation of the
dentin-pulp complex defense reactions by stimulating odontoblast differentiation
and reparative dentin formation (Smith 2003, Tziafas 2004).
2.3.1 Inflammatory cytokines in the dentin-pulp complex
Multiple inflammatory cytokines have been detected in the dentin-pulp complex

of healthy and/or diseased teeth (Table 1).
25
Table 1. Inflammatory cytokines in the dentin-pulp complex, their predominant
functions and targets.
Cytokine Function Target

CCL2 chemokine iDC, monocyte, T cell, etc.
CCL7 chemokine monocyte, NK cell, basophil, etc.
CCL20 chemokine iDC, T cell, B Cell
CCL26 chemokine eosinophil, basophil, iDC, etc
G-CSF action and differentiation granulocyte
M-CSF action and differentiation macrophage
GM-CSF action and differentiation granulocyte, macrophage
CXCL2 chemokine neutrophil
CXCL4 chemokine neutrophil, fibroblast
CXCL5 chemokine neutrophil
CXCL10 chemokine NK cells
CXCL11 chemokine T cell, monocyte, granulocyte
CXCL12 chemokine widespread
CXCL14 chemokine unknown
IFN- proinflammatory phagocyte, T Cell
IL-1 proinflammatory T cell proliferation, indirect
stimulation of chemokines
IL-1 proinflammatory T cell proliferation, indirect
stimulation of chemokines
IL-2 proinflammatory T cell, B cell, NK
IL-3 (multi-CSF) chemokine eosinophil, B cell
IL-4 anti-inflammatory T cell, B cell, mast cell
IL-6 pro/anti-inflammatory T cell, B cell
IL-8 (CXCL8) chemokine neutrophil
IL-10 anti-inflammatory T cell
IL-18 proinflammatory multifunctional
TGF-1 pro/anti-inflammatory, chemokine multifunctional
TNF- proinflammatory multifunctional
Inflammatory cytokines in healthy pulp
Various ILs, which are a group of cytokines with multiple pro- and anti-
inflammatory functions, including chemotaxis, proliferation and stimulation of
various leukocytes and suppression of other cytokines, are expressed in pulp
tissue. IL-1, -1, -2, -4, -6, -8, -10 and IL-18 have all been detected in healthy
human pulp in vivo (Zehnder et al. 2003, Anderson et al. 2002, Hahn et al. 2000).
26
Likewise, multiple ILs, including IL-1, -1, -2, -6, have been observed in
healthy murine pulp tissue in vivo (Bletsa et al. 2004, Bletsa et al. 2006).
Low amounts of IFN- and TNF- have also been detected in human and rat
pulp tissue in vivo (Hahn et al. 2000, Bletsa et al. 2006, Tani-Ishii et al. 1995).
TNF- is a multifunctional proinflammatory cytokine secreted by several cell
types but predominantly by monocytes/macrophages, with additional effects on
lipid metabolism, coagulation, insulin resistance, and endothelial function
(Feghali & Wright 1997). Proinflammatory cytokine IFN- is produced by
activated T-lymphocytes and natural killer (NK) cells and enhances the function
of phagocytes (Feghali & Wright 1997, Vilcek 2002). Additionally, the expression
of granulocyte colony stimulating factor (G-CSF) by untreated human pulp
fibroblasts in vitro has been observed (Sawa et al. 2003). Colony-stimulating
factors (CSFs), granulocyte(G)-CSF, macrophage(M)-CSF, granulocyte-
macrophage(GM)-CSF and multifunctional (multi)-CSF (also known as IL-3) are
cytokines necessary for the survival, proliferation and differentiation of
hematopoietic progenitor cells as well as for the overall immune competence
(Barreda et al. 2004). Chemokines CCL2, CCL7, CCL26, CXCL2, CXCL12 and
CXCL14 have been detected in untreated cultured pulp fibroblasts in vitro
(Staquet et al. 2008).
Inflammatory cytokines in healthy odontoblasts
Due to their location in the periphery of pulp tissue, odontoblasts are the first cells
in the dentin-pulp complex to encounter the bacteria or bacterial products that
penetrate through the enamel and the dentin (Vongsavan et al. 2000). The
odontoblasts constitutively express low levels of IL-8 (also known as CXCL8)
and other chemokines including CCL2, CCL20, CCL26, CXCL4, CXCL12 and
CXCL14, as well as chemokine receptors CXCR2, CCRL1 and CCRL2
(Veerayutthwilai et al. 2007, Levin et al. 1999, Staquet et al. 2008, Durand et al.
2006). CCL2, CXCL12 and CXCL14 are known to attract immature dendritic
cells, while CCL26 functions in the suppression of their recruitment. TGF-1 is
secreted by odontoblasts in healthy teeth, and its expression increases in
irreversible pulpitis (Sloan et al. 2000, Piattelli et al. 2004). Nonetheless, the
knowledge of the odontoblast cytokine expression profile remains restricted.
27
2.3.2 Cytokine response of the dentin-pulp complex to caries
For inflamed pulp to survive, it is essential that the infection is removed and the
inflammation is controlled. For these functions, both pro- and anti-inflammatory
cytokines are needed. In the inflammatory response, exogenous factors, e.g.
bacterial products, cause induction of cytokines that activate the immune response,
while chemokines function as mediators to recruit various leukocytes. Cytokines
interact and function in a synergistic or antagonistic fashion, defining the type of
cells that infiltrate the site of inflammation and the outcome of the disease.
(Gouwy et al. 2005). The functions of specific cytokines in various immune and
inflammatory conditions, including caries, are not well understood due to intricate
and overlapping functions of the inflammatory cytokines.
During caries, bacterial antigens diffuse through the dentinal tubules into the
pulp, leading to the activation of the immune system and inflammation (Smith
2003, Trowbridge 2002, Hahn & Liewehr 2007). Both gram-positive and gram-
negative bacteria are present in the carious lesion, and thus lipoteichoic acid
(LTA), produced by gram-positive bacteria, and lipopolysaccharides (LPS), a cell
wall component of the gram-negative bacteria, have been used to induce
inflammatory reaction in the dentin-pulp complex in experimental conditions
(Hahn & Liewehr 2007). Both LPS and LTA bind to Toll-like receptors (TLRs)
that play critical roles in the recognition of microbial components and trigger
innate and adaptive immunity (Verstak et al. 2007, Takeda & Akira 2005). The
activation of TLRs induces the production of proinflammatory cytokines, such as
TNF-, IL-1, IL-8, IL-12, and also anti-inflammatory cytokines, e.g. IL-10
(Henderson & Wilson 1996, Ginsburg 2002).
Cytokine response of pulp to caries
The expression of various inflammatory cytokines, e.g. several ILs and

chemokines, has been observed in inflamed human pulp tissue in vivo (Zehnder et
al. 2003, Hahn et al. 2000, McLachlan et al. 2004, Takahashi et al. 2008), while
an abundant number of IL-1 and TNF- producing cells, primarily fibroblasts
and macrophages as well as some neutrophils, have been observed in inflamed
pulp tissue of rat (Tani-Ishii et al. 1995) (Table 2).
After LPS treatment of rat pulp in vivo, increased gene expression and
elevated amounts of several proinflammatory cytokines have been observed in
interstitial fluid (Kawashima et al. 2005, Bletsa et al. 2006) (Table 2). Also, in
28
vitro stimulation of cultured human pulp fibroblasts with LPS and another
bacterial product methyl mercaptan have been shown to induce IL-6 production
(Coil et al. 2004, Tokuda et al. 2001). Additionally, endodontic pathogens have
been shown to stimulate IL-6 and -8 production in cultured human pulp
fibroblasts in vitro (Yang et al. 2003a, Yang et al. 2003b). LPS treatment of pulp
fibroblasts in vitro also upregulates the gene expression of several chemokines,
including CCL2, CCL7, CCL26, CXCL10 and CXCL11, while LTA treatment
upregulates CCL2 and -7 (Staquet et al. 2008) (Table 2).
Table 2. Cytokines upregulated in inflamed pulp tissue in vivo or in response to

treatment with inflammatory reaction triggering agents in vitro.
Cytokine In vivo In vitro Reference

(animal) (animal/treatment)
CCL2 human/LPS, LTA Staquet et al. 2008
CCL7 human/LPS, LTA Staquet et al. 2008
CCL20 human human/IL-1, TNF- Takahashi et al. 2008
CCL26 human/LPS Staquet et al. 2008
CXCL5 human McLachlan et al. 2004,
CXCL10 human/LPS Staquet et al. 2008
CXCL11 human/LPS Staquet et al. 2008
GM-CSF human human/TNF- Sawa et al. 2003
M-CSF human human/TNF- Sawa et al. 2003
IFN- human, rat Hahn et al. 2000, Bletsa et al. 2006
IL-1 rat Tani-Ishii et al. 1995, Kawashima et al. 2005, Bletsa et
al. 2006
IL-1 human, rat McLachlan et al. 2004, Takahashi et al. 2008,
Kawashima et al. 2005, Bletsa et al. 2006
IL-4 human Hahn et al. 2000
IL-6 human, rat human/pathogen McLachlan et al. 2004, Zehnder et al. 2003, Bletsa et al.
2006, Yang et al. 2003a, Yang et al. 2003b
IL-8 human human/IL-1, TNF-, McLachlan et al. 2004, Takahashi et al. 2008, Zehnder et
pathogen al. 2003, Yang et al. 2003a, Yang et al. 2003b
IL-10 human, rat Hahn et al. 2000, Kawashima et al. 2005
IL-18 human Zehnder et al. 2003
TNF- human, rat McLachlan et al. 2004, Kawashima et al. 2005, Bletsa et
al. 2006
29
Cytokine response of odontoblasts to caries
Bacterial antigens induce the production of proinflammatory cytokines by

odontoblasts (Hahn & Liewehr 2007). Several proinflammatory cytokines have
been observed in the odontoblast layer of carious human teeth in vivo (Sawa et al.
2003, Durand et al. 2006, Veerayutthwilai et al. 2007) (Table 3).
Odontoblasts demonstrate a differential cytokine response to the antigens of
gram-positive and gram-negative bacteria in vitro (Veerayutthwilai et al. 2007).
LPS, a component of the cell wall of gram-negative bacteria, stimulates human
odontoblasts to produce cytokines IL-1, -8 (CXCL8), TNF-, and CCL20 as
well as CCR6, a receptor of CCL20, in vitro (Veerayutthwilai et al. 2007, Levin et
al. 1999) (Table 3). The stimulation of odontoblasts in vitro with TLR2 ligand,
mimicking the effect of gram-positive bacteria, causes downregulation of the
cytokines upregulated by LPS (Veerayutthwilai et al. 2007). On the other hand,
LTA, a component of gram-positive bacteria, stimulates the gene expression of
chemokines CCL2, CCL7, CXCL2 and CXCL10 in odontoblasts in vitro (Staquet
et al. 2008, Durand et al. 2006) (Table 3). All these chemokines contribute to the
recruitment of different leukocytes (Mantovani et al. 2004). Also, the gene
expression of TGF-1 is down-regulated in odontoblasts challenged with LTA in
vitro (Durand et al. 2006).
Table 3. Cytokines upregulated in inflamed odontoblasts in vivo or in response to

treatment with inflammatory reaction triggering agents in vitro.
Cytokine In vivo In vitro Reference

(animal) (animal/treatment)
CCL2 human human/LTA Durand et al. 2006, Staquet et al. 2008
CCL7 human/LTA Durand et al. 2006,Staquet et al. 2008
CCL20 human human/LPS Veerayutthwilai et al. 2007
CXCL2 human/LTA Durand et al. 2006, Staquet et al. 2008
CXCL10 human/LTA Durand et al. 2006, Staquet et al. 2008
G-CSF human Sawa et al. 2003
M-CSF human Sawa et al. 2003
IL-1 human human/LPS Veerayutthwilai et al. 2007
IL-8 human/LPS Levin et al. 1999, Veerayutthwilai et al. 2007
TNF- human human/LPS Veerayutthwilai et al. 2007
30
2.4 Neuropeptides in the dentin-pulp complex
Neuropeptides are peptides secreted by neurons and function as signaling

molecules either at a synapse or elsewhere (Lodish et al. 2000). In teeth
neuropeptides play roles in vasoactive functions (Heyeraas & Berggreen 1999,
Berggreen & Heyeraas 2000), regulation of inflammatory reactions (Fristad et al.
1997) and proliferation of pulpal cells (Bongenhielm et al. 1995). Several
neuropeptides, including substance P, CGRP, neurokinin A, neuropeptide K,
somatostatin and vasoactive interstitial peptide, have been detected in pulp tissue
(Fouad 2002). The nerve fibers extending into the dentin contain neuropeptides,
including CGRP and substance P (Silverman & Kruger 1987, Wakisaka et al.
1985, Byers & Nrhi 1999). The nerve endings form varicosities near the
odontoblasts, and adhesion-like contacts between the odontoblasts and nerve
endings have been observed (Byers 1984, Byers & Nrhi 1999). The intradentinal
nerve endings are capable of exocytosis and are thought to be able to mediate
efferent signals (Norlin et al. 1999).
Several neuropeptide receptors have been observed in odontoblasts.
Neurokinin 1 (NK1) receptor, which has a high affinity to substance P, and NK2
receptors have been detected in rat odontoblasts (Kido et al. 2005, Fristad et al.
2003), the NK1 amount being higher in the odontoblast cell process than in the
cell body (Kido et al. 2005). The receptor for CGRP has not been detected in
odontoblasts (Fristad et al. 2003). The neuropeptide bradykinin has been shown
to control phosphorylation of endothelial nitric oxide synthase by odontoblasts
(Korkmaz et al. 2006). With the exception of neurotensin (NT), there are no
reports of neuropeptide expression by odontoblasts. NT immunoreactivity has
been observed in rat odontoblasts and has been indicated in the nociception of
tooth (Bhatnagar et al. 1995).
2.5 Transcriptome and proteome of the dentin-pulp complex
The human genome consists of approximately 3 billion nucleotide base pairs, and
the current estimates of the total number of genes vary between 20000 and 25000
(Human Genome Project, http://www.ornl.gov/sci/techresources/
Human_Genome/home.shtml). While the genetic information, the genome, is the
same in every cell, the mRNA and protein content, the transcriptome and the
proteome, vary between the cells. The number of genes expressed in a cell
depends on the environmental and developmental conditions. In general, only a
31
fraction of the genes are expressed at a certain time in any cell type or tissue.
Thus, gene expression studies are used to describe the abundance of certain
mRNA molecules produced in a cell or a collection of cells at a given time or in
given circumstances. Protein levels reflect the ongoing biological and functional
processes of the cell. However, there is no reliable correlation between gene
activity and protein amount (Anderson & Seilhamer 1997). There are several
reasons for this, including the degradation or inefficient translation of mRNA.
The major advance of proteomics, compared to transcriptomics, is that it enables
the detection of protein isoforms resulting from post-translational modifications,
proteolytic processing, or alternative mRNA splicing.
Traditionally, gene and protein expression studies have been performed one
gene or protein at a time. Various methods, such as different PCR techniques, in
situ hybridization, Northern blot at the mRNA level as well as Western blot and
immunohistochemical staining at the protein level, have been used to study gene
and protein expression. All these methods have the disadvantage of enabling the
expression analysis of only a limited amount of genes or proteins simultaneously.
In recent years, the use of methods enabling large-scale expression analysis
of transcriptomes and proteomes has rapidly increased in molecular biology and
biomedical science. These methods enable the expression of thousands of genes
and proteins to be studied simultaneously and thus enable the formation of
genome-wide expression profiles. Large-scale screening methods are also
powerful tools to discover new genes, and to search for interestingly behaving
genes and proteins, which could be used as biomarkers for diagnosis and
estimation of pulp tissue defense potential, and potential target proteins for
treatment. Even though the large-scale expression analysis techniques are widely
used in biomedical research, they have been used only rarely in pulp biology.
2.5.1 Transcriptomics
High-throughput methods to study transcriptome include e.g. DNA microarray,

differential display, serial analysis of gene expression (SAGE), massive parallel
signature sequencing (MPSS), and suppression subtractive hybridization (SSH).
DNA Microarray
A DNA microarray consists of oligonucleotides or cDNA fragments that are either

synthesized directly or spotted onto a solid support, such as a glass slide or filter.
32
The arrays are designed to simultaneously measure the expression level of all
genes represented on the array, and thus allow the simultaneous expression
analysis of thousands of genes in a particular tissue or cell type. The basic
concept of microarray technology is to hybridize test oligonucleotides prepared
from the studied mRNA sample (called targets) to the complementary sequences
called probes, which are bound to a solid surface.
The two most commonly used microarray types are based on a collection of
cDNA fragments (Schena et al. 1995) or short (25mer) oligonucleotides produced
by photolithography (Affymetrix GeneChip) (Lockhart et al. 1996). In addition,
microarrays containing longer (60100 bp) oligonucleotide probes, which are
produced by an inkjet printing process (Agilent arrays), can be used. There are
also different labeling methods. Traditionally, single- or double-labeling is used.
Recently, a method using a third label for quality control purposes has been
introduced (Wang et al. 2003, Wang et al. 2006). When double-labeling is used,
mRNA from two different samples is converted into cDNA and labeled with two
different fluorescent dyes. The samples (targets) are then mixed and hybridized
onto the same array, resulting in competitive binding of the targets to the probe
sequences (Fig. 2.). The array is then scanned at a suitable wavelength to produce
a composite image. The use of double-labeling reduces the effect of slight
differences between the arrays. In the system used by Affymetrix GeneChip
arrays, mRNA is reverse transcribed to produce double-stranded cDNA, which is
in vitro transcribed and amplified into biotin-labeled cRNA. The cRNA is then
fragmented and hybridized onto the arrays. After several washes the cRNA is
stained with streptavidin-phycoerythrin and the array is scanned (Fig. 2.)
The high density oligonucleotide array manufactured by Affymetrix is the
most commonly used microarray platform. Of the ten most used platforms in the
Gene Expression Ontology (GEO) database, nine are manufactured by
Affymetrix, Inc. The Affymetrix microarrays have been proven highly
reproducible (Lockhart et al. 1996) and the Affymetrix system includes its own
software for scanning the array slides and raw data analysis of the slides.
33
Fig. 2. Schematic figure of target preparation for microarray analysis using double-
labeling or the single-label system used by Affymetrix GeneChip.
In any microarray experiment image analysis, signal adjustment and data

normalization to correct for the non-biological variability (or noise) is required.
After this processing, the normalized gene expression data can be analyzed using
various statistical tools and exploratory methods to extract genes or patterns with
biological significance. Thus, the data analysis of a microarray experiment is a
multi-step process, and various analysis programs have been developed.
34
Since DNA microarray enables the monitoring of gene expression of tens of
thousands of genes in several samples in one experiment, immense amounts of
data are produced. Primary microarray data are expensive and time-consuming to
collect, but despite the high cost, the experiments are rarely fully mined. Thus,
several databases, such as Gene Expression Ontology (GEO), ArrayExpress
(AEX) and Celsius, which function as repositories of microarray data, have been
formed. Several meta-analyses using archived data have been published (Rhodes
et al. 2004, Stuart et al. 2003) demonstrating the benefits of these databases.
Due to the wide selection of methods and applications for production and
normalization of microarray data, comparability of the analysis results of
microarray experiments is a major challenge. For this purpose, guidelines for
reporting microarray data and standards for minimum information about
microarray experiments (MIAME) (Brazma et al. 2001) have been developed by
the Microarray Gene Expression Data Society (MGED). Compliance with
MIAME standards and deposition of the original datasets to a public database are,
at present, considered a requirement for publication of microarray data by many
journals. MIAME includes standardized guidelines for presenting information of
both array design and the gene expression experiment.
Suppression subtractive hybridization (SSH)
The SSH process normalizes the levels of rare and abundant genes, and subtracts
genes expressed in both samples, and can thus be used to find genes expressed in
one sample but not in the other. The method can be generally divided into six
steps: (1) synthesis of tester/driver cDNAs; (2) digestion by a four-base cutting
restrictive enzyme yielding blunt ends; (3) separation of the tester cDNA into two
samples, followed by ligation with two different suppression-adapter; (4) two
successive subtractive hybridization; (5) PCR amplification of target sequences
recognized by the different adapters; and (6) construction of the subtracted library
(Diatchenko et al. 1996). The main advantage of SSH in comparison with the
microarray techniques is that SSH does not require any sequence information
prior analysis.
Serial analysis of gene expression (SAGE)
SAGE (Velculescu et al. 1995) is based on the fact that a nucleotide sequence as
short as 910 base pairs provides enough information for identification of a
35
transcript. In SAGE, cDNA is cleaved with restriction enzymes and the 3' ends
are attached to streptavidin beads. The cDNA is then divided into two parts and
ligated with different primers, including a recognition site for a restriction
endonuclease cleaving around 20 base pairs away from the recognition site. This
leads to small tags of cDNAs, which can be ligated together and cloned in
tandem. SAGE provides both qualitative and quantitative information of gene
expression levels.
Massive parallel signature sequencing (MPSS)
MPSS (Brenner et al. 2000) is a powerful sequencing method generating millions

of sequences simultaneously using microbeads. In MPSS, the 3' ends of a cDNA
library are cloned in vitro on microbeads, from which 20 bp long sequences,
generated for each transcript, are sequenced. An advantage of MPSS, in
comparison with other sequencing-based methods (e.g. SAGE), is that DNA
fragment separation is not necessary. With MPSS, more than 500 000 signatures
can be sequenced in a single run. The number of signatures directly reflects the
expression level of the corresponding gene. To identify the transcripts, the
signatures are matched to the genomic sequence.
2.5.2 Proteomics
Several methods can be used for large-scale proteome studies, including 2-D
electrophoresis or liquid chromatography, both usually combined with mass
spectrometry (MS), antibody-based protein arrays, and biomolecular interaction
analysis/mass spectrometry (BIA/MS). The protein separation with 2-D
electrophoresis and subsequent identification with MS are the most commonly
used methods for large-scale proteomics (Bertone & Snyder 2005).
2-D electrophoresis
In 2-D electrophoresis, proteins are first separated in one direction by isoelectric

focusing (IEF) and then in the second direction by molecular mass using
electrophoresis in a slab gel containing sodium dodecyl sulfate (SDS) (OFarrell
et al. 1977). With this method several thousand proteins can be separated, making
it one of the most powerful tools for proteomics. However, 2-D gels are laborious
and time-consuming to run and their dynamic range is limited, while their ability
36
to detect weakly soluble or rare proteins is poor. Additionally, 2-D
electrophoresis provides only means for the separation of proteins, which then
have to be identified by e.g. MS. (Zhu et al. 2003)
Antibody arrays
In antibody-based arrays antibodies are spotted onto a surface, such as a glass

slide, and e.g. a cell lysate or serum is passed over the surface to allow the sample
antigens to bind to the specific antibodies. The bound antigen is then detected by
using fluorescently tagged or radioactively labeled proteins, or by using
secondary antibodies against each antigen of interest (Zhu et al. 2003). The
biggest problem of antibody arrays is the specificity of the antibodies.
Nonetheless, there is evidence that antibody arrays can be used for detection of
antigen abundance in complex mixtures such as serum (Haab et al. 2001, Miller
et al. 2001). When compared with 2-D electrophoresis, the antibody arrays have
the clear advantage of being able to detect very small protein quantities.
Biomolecular interaction analysis/mass spectrometry (BIA/MS)
BIA/MS is a method in which binding of an analyte in solution to a surface-

bound binding partner is analyzed by monitoring surface plasmon resonance
(SPR), and the bound analytes are identified by MS (Krone et al. 1997). SPR is a
label-free quantification method detecting the interaction of light photons with
free electrons (surface plasmons) on a gold surface to quantify the changes in
concentration or amount of biomaterial on the surface (McDonnell 2001).
BIA/MS is a useful and sensitive analytical tool to confirm and quantify affinities
of proteins (Lopez et al. 2003). However, it requires special equipment and is not
widely used.
2.5.3 Transcriptomics and proteomics in pulp biology
Most studies of the transcriptome and proteome of dental pulp and odontoblasts
have been made using traditional methods that enable studying one or a few genes
or proteins simultaneously.
Microarray techniques have been used in a small number of studies in pulp
biological research. cDNA microarray has been used to compare dental pulp stem
cells with mesenchymal stem cells (Yamada et al. 2006) and with bone marrow
37
stem cells (Shi et al. 2001). The Affymetrix oligonucleotide microarray has also
been used to study gene expression changes caused by severe caries (McLachlan
et al. 2005). Tete et al. (2008) have used DNA microarray to profile the mRNA
expression of individual healthy human pulp tissues. Liu et al. (2007) have used
an oligonucleotide-based microarray to identify genes associated with dental pulp
stem cell mineralization. cDNA microarray has been used to study the effects of
LPS and LTA on TLR and chemokine expression of cultured pulp fibroblasts and
odontoblasts (Staquet et al. 2008, Durand et al. 2006). Chen et al. (2005) used
cDNA microarray to study differential gene expression between pulp cell cultures
established from cleidocranial dysplasia patients and healthy controls.
SSH has been used to compare large-scale gene expression between cultured
fibroblastic pulp cells and cultured odontoblast-like cells (Buchaille et al. 2000).
The same method has been used to search for genes expressed differentially or
predominantly in rat odontoblasts, by subtracting the genes commonly expressed
in odontoblasts, osteoblasts and pulp cells (Dey et al. 2001). Additionally, SSH
has been used to compare cultured odontoblast-like cells and cultured osteoblasts
(Hao et al. 2005).
The use of large-scale proteomics in pulp biological studies has been very
limited. Antibody array has been used e.g. to evaluate the effects of LTA on
chemokine expression by odontoblasts (Durand et al. 2006) and the roles of
odontoblasts and fibroblasts in immunity (Staquet et al. 2008).
38
3 Aims of the study
The specific aims of this study were:
1. To investigate the suitability of large-scale expression analysis methods for
pulp-biological research in vivo and in vitro.
2. To study the effects of caries on the transcriptome and proteome of human
pulp tissue.
3. To investigate the effects of TGF-1 on the transcriptome of pulp tissue and
odontoblasts in vitro. The specific interest was on the expression profile of
interleukins.
4. To search for potential novel odontoblast markers by comparing the
transcriptome of pulp tissue and odontoblasts.
5. To compare the transcriptome of mature native odontoblasts with cultured
newly differentiated odontoblasts to provide insight into the relevance of
odontoblast-like cell culturing to mature human odontoblasts. The specific
interest was on the expression of neuronal proteins.
39
40
4 Materials and methods
4.1 Samples
4.1.1 Collection of mature native teeth and native odontoblast and

pulp samples (IIV)
Intact (total n=179) and carious (n=62) human third molars were obtained for the
study from the Oulu Health Care Center Dental Specialty Care Unit and the
University Student Health Care Center, Oulu. All the teeth were removed during
normal treatment, and the patients were healthy, young (1825 years) adults. The
molars were used for the experiments after the patients informed consent,
following the guidelines of the Faculty of Medicine at the University of Oulu for
the use of human samples in research. The study was approved by the Ethical
Committee of the Northern Ostrobothnia Hospital District (232003). The carious
teeth (I) were affected by enamel caries or early dentinal caries. Teeth with deep
caries were excluded. The teeth were immersed in phosphate-buffered saline
(PBS) and used immediately for the experiments or stored at 70 C.
Native pulp tissue and odontoblast samples (IIV) were obtained as
previously described (Palosaari et al. 2000). Crown and root were separated by
cutting a groove with a diamond-edged disc 23mm apically from the cement-
enamel junction, and the roots were fractured off through the cutting line with
sharp-edged pliers. Next, the pulp tissue was carefully removed with forceps and
either cut to pieces and put into Trizol-reagent (GIBCO-BRL, Gaithersburg, MD,
USA) (I) or frozen with liquid nitrogen and pulverized (IIIII). The pulp chamber
was washed with PBS, filled with Trizol-reagent, and the odontoblasts remaining
on the walls of the pulp chamber were removed and collected with a dental
excavator (IIIV).
Developing human third molars used for culturing odontoblast-like cells (IV)
and for immunofluorescent analyses (IV) were extracted for orthodontic reasons,
and informed consent was obtained from the patients in accordance with French
legal requirements (Article 672-1, Public Health Code).
41
4.1.2 Pulp tissue and odontoblast cultures (II, III)
The pulp tissues and odontoblasts were cultured using method described by
Tjderhane et al. (1998). Briefly, after cutting the roots of healthy third molars,
pulp tissues were gently removed and cultured in serum-free OPTI-MEM 1
(Invitrogen, Paisley, UK) culture medium supplemented with 100 IU/ml
penicillin-streptomycin (Biowhittaker Europe, Verviers, Belgium), 0.25 g/ml
Fungizone (Gibco-BRL, Paisley, UK) and 50 g/ml vitamin C (Sigma, Saint
Louis, MO, USA). To culture odontoblasts, the crowns with odontoblasts
remaining on the walls of the pulp chamber were embedded upside down to
agarose gel supplemented with Dulbeccos modified medium and 0.25 g/ml
Fungizone (Gibco-BRL, Paisley, UK), and the pulp chambers were filled with the
same culture medium as used to culture pulp tissues. Next, the crowns were
covered with glass cover slips to prevent evaporation. Odontoblasts and pulp
tissue were cultured with and without 1 ng/ml of TGF-1 for different periods.
The culturing took place at 37 C in the presence of 5% CO2. After the culture
period, 1 h, 5 h, 24 h or 48 h for pulp tissue and 1h or 24h for odontoblasts, the
pulp tissues were frozen with liquid nitrogen and odontoblasts were scraped with
a dental excavator into Trizol-reagent (GIBCO-BRL, Gaithersburg, MD, USA)
and frozen. The culture media were collected after the culture period and stored at
20 C.
4.1.3 Cultured odontoblast-like cells (IV)
The odontoblast-like cells were cultured as previously described (Couble et al.

2000). Pulp explants were grown in Eagles basal medium (Invitrogen, Grand
Island, NY) supplemented with ascorbic acid, antibiotics, and fetal calf serum.
Sodium -glycerophosphate (10 mM) was added to the medium to differentiate
the cells into odontoblast-like cells as described previously (Couble et al. 2000).
After 23 weeks of culturing, the cells differentiated into odontoblast-like cells
with typical (position of the nucleus, cellular extension, junctional complexes,
intracellular organelles) and functional features of odontoblasts (type I collagen,
dentin sialophosphoprotein, enamelysin, osteoadherin).
42
4.1.4 Mouse bone samples (II)
Twenty tibia of four days old mice (provided by Outi Lahti, Department of
Anatomy and Cell Biology, University of Oulu) were frozen in liquid nitrogen,
pooled and pulverized for the protein extraction.
4.2 Extraction of RNA and proteins (IIV)
Total RNA of pulp tissue and odontoblasts was extracted with Trizol reagent
(GIBCO-BRL, Gaithersburg, MD, USA) and further purified with the Qiagen
RNeasy Mini Kit (Hilden, Germany) according to the manufacturers instructions.
The purity of RNA was determined by measuring absorbances at 260 nm and 280
nm, and only 260 nm/280 nm ratios greater than 1.7 were accepted. RNA integrity
was assessed by agarose gel electrophoresis so that only RNA preparations with
clear 18S and 28S bands were accepted for further analyses. Total protein extracts
were prepared from pulp tissue and odontoblast samples according to the Trizol-
reagent protocol.
Total RNA from odontoblast-like cells was extracted using the Nucleospin
RNA II kit (Macherey-Nagel, Dren, Germany) according to the manufacturers
instructions (IV).
From mouse tibia, proteins were extracted by elution with a buffer containing
50 mM TRIS, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij-35 by incubation for
1 h at 4 C (II).
4.3 Large-scale analysis of gene expression
4.3.1 cDNA microarray (I)
Atlas Glass Human 1.0 microarrays (Clontech Laboratories, Palo Alto, CA, USA)
were used for gene expression analysis between healthy and carious teeth pulp
samples. Pulps of healthy teeth were divided into two pools, referred to below as
h1 (n=8) and h2 (n=10), and those of carious teeth into two pools, referred to as
c1 (n=17) and c2 (n=20). Ten micrograms of RNA from each pool was treated
with 1 U of RQ1 RNase-Free DNase (Promega, Madison, WI, USA) and
fluorescently labeled with Cy5 (carious samples, red label) and Cy3 (healthy
samples, green label) (Amersham Pharmacia Biotech, Buckinghamshire, UK),
respectively, with the Atlas Glass Fluorescent Labeling Kit (Clontech) according
43
to the manufacturers manual. Briefly, RNA was reverse transcribed into cDNA,
into which the aminoallyl dUTP was incorporated. Then, N-hydroxysuccinimide-
activated Cy3 or Cy5 was coupled with the modified UTPs, and the probes were
purified with nucleospin columns. The labeled probes were hybridized to the
microarray chips according to the manufacturers instructions. Briefly, labeled
probes were combined and diluted into hybridization buffer and added to the
hybridization chamber containing the chip. The chamber was incubated overnight
(at least 16 h) at 50C and washed. Pooled samples c1 and h1 were hybridized
once and samples c2 and h2 were hybridized twice.
The microarrays were scanned using a GSI Lumonics SA 5000 scanner with
the ScanArray software (GSI Lumonics, Watertown, MA, USA), and image
analysis was performed with QuantArray software (GSI Lumonics) according to
the manufacturers instructions. The scanned images for both labels were
combined into one composite image, in which the color of each spot represented
the measured Cy3/Cy5 intensity ratio of each gene. The efficiency of cDNA
synthesis and dye incorporation was confirmed by strong and equal intensity of
control spots. All irregularly shaped spots were excluded from further analysis.
Differential expression of genes in healthy vs. diseased teeth was analyzed by
calculating the Cy3/Cy5 intensity ratio for each spot from the intensity values
acquired from the QuantArray software. The mean of ratios for two independent
hybridizations for samples c2 and h2 were calculated. For normalization a single
scaling factor was applied to each chip so that the average Cy3/Cy5 ratio was 1.0
to balance the individual hybridization signals for meaningful comparison. The
cut-off point for high expression was set as at least five times the background
fluorescence. 1.4-fold change was selected as the cut-off point for a true change.
4.3.2 GeneChip microarray (II, III, IV)
GeneChip microarrays (Affymetrix, Santa Clara, CA, USA) were performed

using Affymetrix HGU133A human genome arrays for native and cultured pulp
tissues and using Affymetrix HGU133plus 2.0 arrays for native and cultured
odontoblasts as well as odontoblast-like cells.
Experimental procedures for GeneChip (Affymetrix, Santa Clara, CA, USA)
were performed according to the Affymetrix GeneChip Expression Analysis
Technical Manual. In essence, double-stranded DNA was synthesized using 10 g
of total RNA as the template by means of the Superscript Choice System (Gibco
BRL Life Technologies, Rockville, MD, USA) and T7-(dT)24 primer. The DNA
44
was purified with the GeneChip Sample Cleanup Module (Qiagen, Valencia, CA,
USA). In vitro transcription was performed to produce biotin-labeled cRNA using
a BioArray HighYield RNA Transcription Labeling Kit (Enzo Diagnostics,
Farmingdale, NY, USA) according to the manufacturers instructions.
Biotinylated cRNA was cleaned with the GeneChip Sample Cleanup Module
(Qiagen), fragmented to 35 to 200 nt, and hybridized to the array. After being
washed, the array was stained with streptavidin-phycoerythrin (Molecular Probes,
Eugene, OR, USA). The staining signal was amplified by biotinylated anti-
streptavidin (Vector Laboratories, Burlingame, CA, USA) and a second staining
with streptavidin-phycoerythrin, and then scanned with an HP GeneArray Scanner.
The expression data was analyzed using Affymetrix GeneChip Operating
Software version 1.1.1. Signal intensities of all probe sets were scaled to the
target value of 500. All samples were hybridized once.
Data analysis (II, III, IV)
Raw data were analyzed using Affymetrix software. Absolute calls (present,
marginal and absent) and signal values (relative indicator of mRNA abundance)
for each assayed gene were calculated by Affymetrix algorithms. Probe sets
giving positive signals (present call) either solely in pulp tissue or in odontoblasts
(II) and either only in native odontoblasts or in cultured odontoblast-like cells (IV)
were analyzed with Onto-Express software (http://vortex.cs.wayne.edu) to divide
the probes into categories according to the Gene Ontology (GO) classifications
(http://www.geneontology.org). Some interesting categories were analyzed further
by checking all the probe sets representing genes of those categories to exclude
those represented by several probes with conflicting expression profiles.
Genes with at least an eight-fold change in response to TGF-1 treatment at
least at one time point were manually divided into GO categories
(http://www.geneontology.org) according to their biological function (III).
Affymetrix CEL-files (raw data files) were imported into dChip software
(www.dchip.org), and the software was used according to the software
manufacturers manual (www.dchip.org) to search for gene categories
demonstrating significant changes in response to TGF-1 treatment (III).
Invariant set normalization was used to normalize the arrays to make them
comparable, and a model-based method was used for probe-selection and
computing expression values. The expression levels were attached with standard
errors as measurement accuracy, which were subsequently used to compute 90%
45
confidence intervals of fold changes in two-sample or two-group comparisons.
Genes with a more than two-fold (lower confidence bound) increase or decrease
in expression after TGF-1 treatment were selected for further study and
classified into categories. The p-values for the classified categories were
calculated from the number of genes belonging to the category with changed
expression and the total number of genes belonging to the category.
Data downloaded from Gene Expression Omnibus (II)
The data of expression profiles of two healthy human dental pulp samples
(McLachlan et al. 2005) under number GSE1629 were downloaded from the
GEO database (http://www.ncbi.nlm.nih.gov/projects/geo/) for comparative gene
expression analysis. These samples had been analyzed using the same array
(Affymetrix HGU133A) as our pulp samples, thus enabling the comparisons.
Numbers of probe sets detecting gene expression were compared between the
samples downloaded from the GEO database and our own pulp tissue sample so
that the amounts of probe sets found only in one, in two, and in all three samples
were calculated. Percentage agreement levels between individual samples were
calculated from amounts of probe sets agreeing and disagreeing in the presence or
absence of expression between two samples.
4.4 RT-PCR (I, II, III)
cDNA was synthesized from 1 g of total RNA with AMV reverse transcriptase
(Promega, Madison, WI, USA) according to the manufacturers instructions. The
PCR reaction was carried out in a mixture containing 200 M dNTP (Promega),
300 nM primers, 2 mM MgCl2 and 2.6 U polymerase (Roche Molecular
Biochemicals, Mannheim, Germany) in a total volume of 50 l. The PCR reaction
began with a 2-minute initial denaturation at 94 C; then ten cycles of
denaturation at 94 C for 15 seconds, annealing at an appropriate temperature for
30 seconds and an extension at 72 C for 45 seconds. After the first ten cycles, the
extension time was extended by 5 seconds per each cycle. Gene specific primers
were used as described in (II, III). When nested PCR was necessary, it was
performed similarly using 1 l of the original PCR product as template.
In part I of this work, a slight variation of RT-PCR, coupled one-step RT-PCR
(Aatsinki 1997), was used. RT- and PCR-reactions were performed in the same
reaction tube, at the final concentration of 1.3x Expand HF buffer, 0.8 mM dNTPs,
46
50 ng of both primers, 1 unit of AMV-RT (Promega, Madison, MI, USA) and 2.6
units of Expand High Fidelity PCR system enzyme mix (Roche Molecular
Biochemicals, Mannheim, Germany) in a total volume of 50 l. The RT-reaction
was carried out for 1 hour at 42 C with a final 3 minutes at 95 C to stop the
reaction. Each PCR cycle consisted of 1 minute denaturing at 95 C, annealing of
2 minutes and extension of 10 minutes + 59 seconds/cycle at 72 C.
When semiquantitative RT-PCR was performed (II, III), an appropriate
number of cycles was determined by taking aliquots of PCR product after a
different number of cycles and running them on an agarose gel, and then selecting
the cycle number still at the linear reaction phase. The amounts of PCR products
were quantified by Scion Image software (Scion Corporation, Frederick, MA,
USA) using -actin as the control of the RNA amount.
4.5 Real-time quantitative PCR (IV)
cDNA was synthesized from 1 g of total RNA using AMV reverse transcriptase
(Promega, Madison, WI, USA), and real-time quantitative PCR (qPCR) reactions
for galanin (GAL), pronociceptin (PNOC) and somatostatin receptor 1 (SSTR1)
were performed with an ABI 7700 Sequence Detection System (Applied
Biosystems, Foster City, CA, USA) using TaqMan chemistry. The results were
normalized to 18S RNA. Real-time qPCR for PNOC and PTPRZ was performed
in a Light Cycler instrument (Roche, Meylan, France) with the Fast Start DNA
Master SYBR Green I kit. The results of PNOC and PTPRZ1 were normalized to
cyclophilin A mRNA.
Amounts in native odontoblasts were selected as baseline and compared with
the expression levels in odontoblast-like cells and cultured pulp cells. The
amplifications were performed in triplicates. The specific primers were used as
described in IV.
4.6 Proteomics
4.6.1 2-D electrophoresis and mass spectrometry (MS) (I)
The 2-D gel electrophoresis and MS analyses were performed as earlier described
by Ohlmeier et al. (2004) using proteins extracted from healthy and carious pulp
tissue. Briefly, 100 g of the protein extract was adjusted to the final volume of
47
400 l with urea. Then the proteins were transferred into IPG strips (pH 310,
non linear, 18 cm; Amersham Biosciences, Uppsala, Sweden) overnight by in-gel
rehydration. IEF was carried out at 20 C with the Multiphor II system
(Amersham Biosciences) under paraffin oil for 55 kVh (Buttner et al. 2001).
Before the separation in the second dimension, the strips were equilibrated
according to Grg et al. (1995). The electrophoresis was performed overnight in
polyacrylamide gels with the Ettan DALT II system (Amersham Biosciences) at
12 W per gel and 12 C. At least three 2-D gels were run for each sample. The
gels were silver stained (Blum et al. 1987), and the protein pattern of healthy and
carious tooth pulp tissue was analyzed with the 2-D gel image analysis software
Melanie 3.0 (GeneBio, Geneva, Switzerland). For fluorescence labeling, 10 g of
protein was labeled with Cy3-dye and run according to the manufacturers
protocol (CyDye DIGE Fluor Labelling Kit for Scarce Samples, Amersham
Biosciences).
For protein identification, selected spots were cut from the gel, digested in the
gel, and the peptide fingerprints were analyzed with matrix-assisted laser-
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as
described earlier (Miinalainen et al. 2003). The identification of a protein was
considered acceptable if the peptides (mass tolerance 20 ppm) covered at least
30% of the complete sequence. A sequence coverage between 30% and 20% or a
sequence coverage below 20% for protein fragments was only accepted if at least
three main peaks of the mass spectrum matched with the sequence and the
number of weak-intensity peaks was clearly reduced.
4.6.2 Antibody array (III)
Pools of culture medium of pulp tissues cultured +/- TGF-1 for 24 h and 48 h
were analyzed with the ProteoPlex 16-Well Human Cytokine Array Kit (Novagen,
Nottingham, UK), which allows detection of IL-1, -1, -2, -4, -6, -7, -8, -10 and
-12, as well as TNF-, IFN- and GM-CSF, according to the manufacturers
instructions. Two different dilutions were used to enable quantitative detection of
all cytokines included on the array. Briefly, the samples were diluted into ratios of
1:1 and 1:9 with 2x sample diluent, applied to the wells of array devices, and
incubated for 1 h at room temperature with careful shaking. Then the arrays were
washed with phosphate-buffered saline buffer containing 0.1% (vol/vol) Tween
20 (PBST) and the antibody cocktail was added. Then the arrays were incubated
as before and washed. Next, PBXL-3 was added and the arrays were incubated at
48
room temperature protected from light for 1 h 30 min. Then they were rinsed with
1x Rinse Solution and dried. The arrays were scanned and the raw data were
analyzed by the manufacturer. The effects of TGF-1 on cytokine protein
amounts were calculated relative to control samples cultured without TGF-1.
4.7 Western blot (II)
Western blot was used to confirm the differential matrilin 4 (MATN4) expression
in native pulp tissue and odontoblasts. Protein (10 g) extracted from pools of
human native pulp tissue and odontoblasts as well as mouse bone, which is
known to express high amounts of MATN4 (Klatt et al. 2001), was treated with
2-mercaptoethanol for 3 minutes at 100 C to achieve reducing conditions and run
on a 12% sodium dodecyl sulphate-polyacryleamide gel (SDS-PAGE). The gel
was stained with Coomassie Brilliant Blue to allow detection of albumin and
scanned. The staining was removed by incubation in a solution containing 30%
ethanol and 10% acetic acid. Next, the proteins were transferred to an Immobilon
P membrane (Millipore Corp., Bedford, MA, USA), and the membrane was
handled using ECL Western blot detection reagents and analysis system
(Amersham Biosciences, Buckinghamshire, UK) according to the manufacturers
instructions. The primary antibody used was rabbit polyclonal antibody against
MATN4 (a kind gift from Dr. Raimund Wagener, Cologne, Germany) at a 1:500
dilution at 20 C overnight. Analyses was performed in triplicates.
To enable the comparison of protein amounts between the samples, the
membranes were stripped and reprobed with a monoclonal antibody against
-actin (Novus Biologicals, Littleton, CO, USA) at a 1:2000 dilution according to
the instructions of ECL Western blot detection reagents (Amersham Biosciences).
Scion Image software (Scion Corporation) was used to determine the
intensities of stained albumin as well as immunoreactive MATN4 and -actin
bands, and relative MATN4 amounts against albumin and -actin were calculated.
MATN4/albumin and MATN4/-actin ratios were normalized by giving the ratio
in the mouse bone sample a value of 1. Means and standard deviations of three
separate analyses were calculated.
4.8 Immunofluorescence (IV)
Cryostat sections (10 m) were prepared as described previously (Allard et al.

2000). Developing third molars were used for the analyses. Odontoblasts located
49
near the crown were mature, while odontoblasts located at the apical part of the
developing root were newly differentiated. The sections were incubated with
nociceptin (PNOC) or galanin (GAL) antibodies (dilution 1:500 and 1:100
respectively, Chemicon, Temacula, CA, USA), SSTR1 antibody (dilution 1:100,
Gramsch Laboratories, Schwabhausen, Germany) and cortistatin (CORT)
antibody (dilution 1:100, Phoenix Pharmaceuticals Inc, Belmont, CA, USA).
After rinsing, the sections were incubated with Alexa fluor 488 goat anti-rabbit
IgG (Invitrogen), and the staining was detected by epifluorescence (Olympus
BX50) microscopy. Negative controls were performed by omitting the primary
antibody or by incubating sections with normal rabbit IgG.
50
5 Results
5.1 The effects of caries on the expression profile of pulp tissue (I)
The effects of caries on the gene expression profile and protein content of pulp
tissue were studied with cDNA microarray and with 2-D electrophoresis
combined with MS, respectively.
5.1.1 The effects of caries on the mRNA profile of pulp tissue
As detected with the cDNA microarray used, 396 (36.6%) genes gave a clearly
positive signal in at least one of the healthy or the carious samples. Paired box
protein 3 (PAX3) and phosholipase A2 (PLA2) gene expression levels decreased
in the pulp tissue of carious teeth, while 12 genes, including placental growth
factor (PlGF), teratoma-derived growth factor 1 (TDGF-1), delta-like protein
(DLK), macrophage-stimulating protein (MSP), G protein-coupled receptor
(EDG4), neurogranin (NGR), jagged homolog 2 (JAG2), embryonic
growth/differentiation factor 1 (GDF-1), endothelin 2 (ET-2), oncostatin M
(OSM), duffy blood group antigen (DARC), and stratifin (SFN), were
upregulated in the pulp tissue of carious teeth. Plexin-related protein B3 (PLX B3)
presented different expression patterns with a decrease in one of the carious
samples and an increase in the other one. Variation between the expression
changes in individual samples was observed.
High intensities (over 5-times the background fluorescence) indicating
probably high expression levels of several genes, including PlGF, PAX3, matrix
metalloproteinase 13 (MMP-13), ubiquitin-conjugating enzyme-E2 (UCE-E2),
interleukin 13 (IL-13), nerve growth factor-2 (NGF-2), TEK tyrosine kinase
(TIE-2), tumor necrosis factor receptor-1 (TNFR-1), nuclear factor 90 (NF 90),
transcription elongation factor SII (TEF-SII), and TDGF-1, were detected in the
pulp tissues of both healthy and carious teeth. The detected intensities of UCE-E2,
NGF-2, TNFR-1, NF-90 and TEF-SII varied markedly between the samples.
Variation between the two hybridizations of the same sample was small. RT-PCR
confirmed the expression of PAX3, PL A2, DLK and NGF-2.
51
5.1.2 The effects of caries on the protein profile
After separation by 2-D electrophoresis, approximately 400 protein spots were

detectable by silver staining of pooled samples and 600 by fluorescent staining of
individual pulp samples. However, none of these proteins demonstrated a
reproducible difference between the pulp of healthy and carious teeth (Fig. 3.).
Fig. 3. Typical 2-D gels of healthy and carious pulp tissue. Approximately 600 protein
spots were detected but no reproducible differences between the protein profiles of
healthy and carious pulp tissue could be observed.
From the 2-D gels, 96 proteins within 157 spots were identified by MS
(http://www.biochem.oulu.fi/proteomics/teeth_gel.html). Several proteins were
localized in multiple spots suggesting fragmentation or posttranslational
modifications. One of the identified proteins was a hypothetical, protein (putative
nucleoside diphosphate kinase, H_278C19.3), representing the first evidence for
the existence of this protein. None of the proteins with a corresponding change at
the mRNA level were detected. Probes for 12 of the genes encoding the identified
proteins were present on the array used, but none of these genes had an intensity
of at least five times the background fluorescence.
52
5.2 Repeatability of Affymetrix GeneChip arrays (II)
To determine the repeatability of the Affymetrix microarrays, expression profile

data of two healthy pulp samples acquired using the same microarray platform
that was used for pulp tissue in this study (II, III) were downloaded from the GEO
database under number GSE1629, and the similarities between positive probe sets
were calculated. The comparison revealed a similarity of 88.2% between the
expression profiles of the two samples downloaded from the GEO database and a
similarity of 85.3% between the samples downloaded from the GEO database and
our own pulp sample.

tissue and odontoblasts (III)
Microarray analysis of native and +/- TGF-1 cultured pulp tissues and
odontoblasts revealed that culturing in itself caused many changes in the gene
expression profiles of pulp and odontoblasts (Table 4). Culturing for short periods
(1 h and 5 h) caused slighter changes than culturing for longer periods (24 h and
48 h) (Table 4). However, TGF-1 caused notable up- or downregulation of many
genes both in pulp tissue and in odontoblasts. The genes, whose expression was
altered at least eight-fold (127 genes in pulp tissue and 122 genes in odontoblasts),
could be distributed into several categories according to their biological function,
including genes related to inflammation, transcription, apoptosis, signaling and
signal transduction, metabolism, cell adhesion, cell cycle and proliferation,
development, protein biosynthesis, modification and transfer, proteolysis, and
different transport mechanisms.
Table 4. Numbers of probe sets detecting over a four-fold expression change in pulp
tissue and odontoblasts (Od) in response to culturing and TGF-1.
Change Culturing TGF-1

1h 5h 24h 48h 1h 5h 24h 48h
Increased in pulp 258 194 800 981 315 96 236 89
Decreased in pulp 106 112 860 1003 148 49 50 129
Increased in Od 545 NA 1080 NA 127 NA 178 NA
Decreased in Od 887 NA 672 NA 96 NA 541 NA
NA: Not analyzed
53
dChip analysis revealed that 1 h and 5 h TGF-1 treatment (short-term culturing)
caused similar changes in the expression profiles of pulp tissues, while the
expression profiles of pulp tissues cultured for 24 h and 48 h (long-term culturing)
differed more from each other and from the earlier time points. Significant
(p<0.002) changes were observed in 19 GO categories (a total of 249 genes) after
short-term culturing, and in five GO categories (97 genes) after long-term
culturing.
dChip analysis revealed that gene expression changes caused by TGF-1 in
odontoblasts cultured for 1 h and 24 h were very different. Regardless of the
expression changes of individual genes, no categories were significantly changed
in response to TGF-1 treatment at both time points.
5.4 Interleukin profile of pulp tissue and odontoblasts (I, III)
5.4.1 Interleukin profile of native pulp tissue
Probes for 17 ILs , including IL-1, -1, -2, -4, -5, -6, -7, -8, -9, -10, -11, -12,
-12, 13, 14, -15 and -16, were included in the cDNA microarray used to study
pulp tissue of native intact and carious teeth. Only IL-6 and -13 gave positive
signal, and of these, only IL-13 was strongly expressed (detection over 5-times
the background) (Table 5). (I)
The Affymetrix GeneChip array used to study native and +/- TGF-1 treated
pulp tissues included probes for 24 different ILs, and IL-7, -12 and -16 mRNAs
were detected in native pulp tissue (Table 5). The expression of these three ILs
was also confirmed by RT-PCR. (III)
Table 5. Detection of interleukins in native healthy and carious pulp tissue by cDNA
microarray and oligonucleotide-based DNA microarray (I, III).
Interleukin cDNA microarray (I) Oligonucleotide microarray (III)

healthy/carious healthy
Il-6 +/+ -
IL-7 -/- +
IL-12 -/- +
Il-13 +/+ -
Il-16 -/- +
-: expression not detected, +: expression detected
54
5.4.2 Effects of TGF-1 on the interleukin expression profile of pulp
tissue (III)
TGF-1 caused at least a 2-fold increase in the expression of IL-1, -1, -6, -8
and -11 after 1 h and 5 h of culturing, while IL-16 and -23 expression was
upregulated at the later time points. By antibody array, IL-1, -1, -2, -4, -6, -7, -8,
-10, and -12 proteins were detected in the culture medium of pulp tissues cultured
for 24 h and 48 h at at least one time point (Table 6). Protein levels of IL-6 and -8
were markedly higher than other cytokine levels detected. TGF-1 caused at least
a 2-fold increase in proinflammatory IL-1, -1, -2, -6, -7, and -12 proteins. No
marked changes were detected in the expression of anti-inflammatory IL-4 or -10
proteins. IL-4 showed no change at 24 h and a 1.4-times increase at 48 h, while
IL-10 was increased 1.8-fold at 24h and decreased 1.3-fold at 48h.
Table 6. Detection of interleukins in cultured pulp tissue by DNA microarray and

antibody array (III).
Interleukin DNA microarray Antibody array

Cultured control Cultured TGF-1 Cultured control Cultured TGF- 1
1h 5h 24h 48h 1h 5h 24h 48h 24h 48h 24h 48h
IL-1 - - - - + + - + + + + +
IL-1 - + - - + + - - + + + +
IL-2 - - - - - - - - - - + -
IL-4 - - - - - - - - + + + +
IL-6 - - + + + + + + ++ ++ ++ ++
IL-7 + + + + + + - - - - + +
IL-8 + - + + + + + + ++ ++ ++ ++
IL-10 - - - - - - - - + + + +
IL-11 - - + + + + + + NA NA NA NA
IL-12 - - + - + + - - + - + +
IL-16 + + + - + + + - NA NA NA NA
IL-23 + - + + - - + + NA NA NA NA
IL-24 - - - + - - - - NA NA NA NA
- :expression not detected, + :expression detected, ++ :very high expression detected by antibody array
NA: not analyzed
55
5.4.3 Interleukin profile of native odontoblasts (III)
IL-6 and 7 were detected in native and all cultured odontoblast samples by
microarray analysis, and the presence of the mRNAs in native odontoblasts was
confirmed by semiquantitative RT-PCR.
5.4.4 Effect of TGF-1 on the interleukin expression profile of

odontoblasts (III)
IL-1 mRNA was detected in odontoblasts cultured both with and without
TGF-1 for 24 h, while IL-8 mRNA was detected in odontoblasts cultured with
TGF-1 for 1 h and 24 h and also in the control sample cultured for 1 h.
IL-8 was the only IL with at least a 2-fold change in response to TGF-1
treatment, with over an 20-fold increase after 1 h and a 2.8-fold decrease after
24 h. Semiquantitative RT-PCR confirmed IL-8 upregulation after 1 h culturing
with TGF-1, while no clear difference in IL-8 mRNA levels could be detected
between samples cultured with and without TGF-1 for 24 h.
5.5 Differences between the general expression profiles of native

pulp tissue and odontoblasts (II)
Comparison of microarray data of native pulp tissue and odontoblasts revealed

9719 positive probe sets (43.7% of the total number of probe sets in the
microarray used for pulp sample) both in pulp tissue and odontoblasts. In addition,
the analyses revealed 1595 positive probe sets (7.2% of the total number of probe
sets) in pulp but not in odontoblasts, and 904 positive probe sets (4.1%) only in
odontoblasts. Probe sets detecting gene expression only in pulp tissue or in
odontoblasts could be divided into several GO categories, including cell
differentiation, ECM organization and biogenesis, apoptosis, cell adhesion, cell-
cell signaling, etc. The distribution of positive probe sets into the GO categories
was very similar between pulp tissue and odontoblasts.
56
5.6 Potential odontoblast markers (II, III)
5.6.1 Matrilin 4 and collagen type XI 1
Only two ECM organization and biogenesis related genes, MATN4 and
COL11A1, were detected in native odontoblasts but not in native pulp tissue by
microarray. Analysis of the expression profiles of two native pulp samples stored
in the GEO database under number GSE1629 revealed COL11A1 expression but
no MATN4 expression. RT-PCR and Western blot confirmed MATN4 expression
only in odontoblasts at the mRNA and protein levels, respectively. Culturing pulp
tissue for 48 h with TGF-1 induced MATN4 expression (Table 7). RT-PCR also
showed the expression of the 354-bp COL11A1 splicing variant in odontoblasts.
However, in pulp tissue the expression of 354-bp and 608-bp variants of
COL11A1 was observed. Relative comparison to -actin revealed that the amount
of the 354-bp variant expressed in both pulp tissue and odontoblasts was in pulp
tissue only approximately one third of the amount observed in odontoblasts.
5.6.2 Expressed sequence tags (EST)
Microarray revealed 16 expressed sequence taqs (EST) and other unclassified

transcripts in native odontoblasts but not in native or cultured pulp tissue (Table
7). Two of these 16 transcripts were consistently expressed in all studied native
and cultured odontoblast samples.
5.6.3 Dentin sialophosphoprotein (DSPP)
Dentin sialophosphoprotein (DSPP), traditionally considered to be odontoblast-

specific, was detected in both native odontoblasts and native pulp tissue as well as
in cultured odontoblasts and pulp tissue with microarray (Table 7).
57
Table 7. The detection of possible new odontoblast markers and DSPP by microarray
in native and cultured odontoblasts and pulp tissue
mRNA Native Od 1h cultured Od 24h cultured Od Pulp tissue

Control TGF-1 Control TGF-1
MATN4 + 48h +TGF
AA071454 + + +
AA521034 + + + +
AA601208 + + + + +
AB020695 + + + +
AF052117 +
AF070571 + + +
AI740515 + + + + +
AK021987 + + +
AK025194 +
AL049278 + + +
AL049280 + +
AL050043 + +
AL109705 + +
AW969803 + + +
L34409 +
U79300 + + +
DSPP + + + + + every sample
+ indicates detected expression, indicates that expression was not detected.
5.7 Comparison of the general expression profiles of mature native

odontoblasts and newly differentiated cultured odontoblast-like
cells (IV)
Microarray analysis of mature native odontoblasts and newly differentiated

cultured odontoblast-like cells revealed 17106 positive probe sets (31.3% of the
total number of probe sets) in both native and cultured samples (Fig. 4.). In
addition, the analysis revealed 3714 (6.7%) probe sets detecting expression only
in mature native odontoblasts and 4937 probe sets (8.9%) only in cultured
odontoblast-like cells. The distribution of probe sets positive only in one of the
samples in the GO categories was very similar between the samples. An over two-
fold difference in the amounts of positive probe sets was observed in only three
categories, including cell cycle, cell growth, which held more probe sets in
cultured sample, and detection of stimulus that was more abundant in native
odontoblasts.
58
Fig. 4. Venn diagram showing the numbers of probe sets detecting gene expression
either in native or in cultured odontoblasts (Od) or in both (overlapping circles).
Percentages indicate numbers of positive probe sets of the total amount of the probe
sets.
5.7.1 Expression of selected neuronal proteins in mature and newly

differentiated odontoblasts
CORT and PTPRZ1 mRNAs were detected in both mature native and cultured
newly differentiated odontoblasts using microarray and qPCR (Table 8). CORT
and PTPRZ1 expression levels detected with qPCR were slightly lower in
cultured odontoblasts than in native odontoblasts. By microarray SSTR1 and
GAL mRNAs were detected in native odontoblasts but not in cultured
odontoblasts (Table 8). By qPCR they were detected in both samples; however,
SSTR1 and GAL expression levels were markedly higher in native odontoblasts
than in cultured odontoblasts. Microarray showed PNOC expression in cultured
odontoblasts but not in native odontoblasts (Table 8). With qPCR, PNOC was
detected in both samples, but PNOC mRNA levels were markedly higher in
cultured than in native odontoblasts.
59
Table 8. Detection of CORT, PTPRZ1, SSTR1, GAL and PNOC mRNA in mature native
and cultured newly differentiated odontoblasts.
mRNA Microarray qPCR

mature/newly differentiated mature/newly differentiated
CORT +/+ +/+
GAL +/- +/+
SSTR1 +/- +/+
PTPRZ1 +/+ +/+
PNOC -/+ +/+
-: expression not detected, +: expression detected
Immunofluorescent analysis of developing tooth revealed CORT protein in newly

differentiated native odontoblasts close to the apical part of the developing root
and in mature odontoblasts located at the crown. GAL was moderately present in
young odontoblasts and highly expressed in mature odontoblasts. Moderate
PNOC staining was detected in young odontoblasts and a lower PNOC level in
mature odontoblasts, with accumulation at the terminal web and odontoblast
processes. SSTR1 staining was not observed in young odontoblasts, but in mature
odontoblasts moderate staining was detected.
60
6 Discussion
In recent years DNA microarray and other high-throughput expression analysis
methods have become widespread in biomedicine. However, the use of these
methods in pulp biological research has been very limited. At the mRNA level,
the DNA microarray is the most powerful screening tool currently available.
DNA microarray experiments have several sources of inaccuracy and
inconsistencies, ranging from technical errors and variable experimental settings
to biological variation. However, good intra- and inter-platform reproducibility
between expression measurements performed in different laboratories has been
reported by the MAQC project (MAQC Consortium et al. 2006, Canales et al.
2006). Also, the high reproducibility of Affymetrix GeneChip arrays has been
shown previously (Lockhart et al. 1996). Microarray data of healthy native pulp
tissue were compared with the expression profile data of two healthy pulp
samples acquired using the same Affymetrix HGU133A microarray and
downloaded from the GEO database (II). This demonstrated high similarity
considering that the microarray analyses were performed using independent
human samples in separate laboratories. Thus, the high repeatability of
Affymetrix microarrays, even between different laboratories, was indicated also
for human pulp samples. A slight variation in the results can be caused even by
different personnel, who are handling identical samples while using the same
methodology and the same equipment (J. Vuoristo, personal communication).
Sample pooling is sometimes used in microarray experiments to reduce the
effect of biological variation and also to generate sufficient RNA amounts when
the available sample is restricted. Pooling has been suggested as an error source in
microarray experiments (Shih et al. 2004). However, there is evidence that
identification of most of the differentially expressed genes is not affected by
pooling (Kendziorski et al. 2005, Mary-Huard et al. 2007). All the samples used
in parts IIV of this study were pooled, but since mRNA amounts acquired from
pulp tissue and especially from the odontoblasts of one human third molar are
very limited and biological variation between the teeth is high, the sample pooling
was deemed beneficial.
DNA microarray does not directly measure gene expression but signal
intensity. Thus, the quality of probes significantly affects results, and they must be
carefully designed. In Affymetrix GeneChip arrays, multiple probes are designed
to bind in the proximity of the 3' end of the transcripts. Thus, detection of
isoforms truncated from 3' end is impossible. Recently, arrays in which probes
61
cover the whole length of the target sequence (e.g. Affymetrix exon arrays) have
been developed to circumvent this problem (Abdueva et al. 2007). An assumption
has been made that the mRNA amounts in cells represent the degree of
transcription of these genes. This is not always true, since transcription and
transcript stability are controlled at many levels, affecting the detected mRNA
levels.
One of the most important factors in a microarray experiment is the data
analysis. For that, several different analysis software using different statistical
tests have been developed. However, a recent report indicates that differentially
expressed genes can be reliably identified by fold-change ranking instead of
relying on statistical significance (MAQC Consortium et al. 2006, Canales et al.
2006). On the other hand, much biologically relevant information can be
extracted from raw microarray data, e.g. by using software designed to find
functionally connected gene groups. For example, in part III of this study, dChip
analysis showed a highly significant change in the category of genes related to
chemokine and cytokine activity, while closer analysis of the fold-changes
revealed that the expression of individual cytokines was altered.
6.1 Suitability of large-scale screening methods in pulp-biological

research
Since the use of large-scale expression screening in pulp biology has been limited,
its suitability for in vivo and in vitro pulp biological studies was investigated.
6.1.1 In vivo analysis of the transcriptome and proteome
The suitability of large-scale screening methods, cDNA microarray and 2-D

electrophoresis, for in vivo research of pulp biology was evaluated by comparison
of the mRNA and protein profiles of pulp tissue of healthy and carious teeth with
these methods. cDNA microarray revealed only a few very slight differences at
the mRNA level and variation between the samples, while with 2-D
electrophoresis no reproducible differences in the protein profiles could be
detected. None of the proteins encoded by the differentially or highly expressed
genes were identified at the protein level; instead, 96 other proteins were detected.
These differences between the observed transcriptome and proteome may reflect
problems caused by both the samples and methods used. Only teeth with small or
moderate caries were used, and thus the pulps of the carious teeth used contained
62
a great amount of healthy tissue (Fig. 5.) that may have diluted the differences.
Also, the ability of 2-D electrophoresis to detect rare or hydrophobic proteins is
restricted (Zhu et al. 2003). Additionally, the cDNA microarray used was able to
detect only a small portion of the transcriptome. It is probable that more gene
expression differences would have been detected by using a microarray platform
detecting a larger set of transcripts.
McLachlan et al. (2005) performed a similar analysis of differences between
healthy and carious pulp tissues detecting various gene expression differences,
most notably genes related to the immune and inflammatory response. However,
since their samples contained teeth with deep lesions, and thus with extensive
pulpal inflammation, the detected expression profile most likely represents
inflammatory cells (Fig. 5.). In contrast, the expression differences between
healthy and carious tissue detected in part I of this study probably represented
dental pulp and not a general inflammatory reaction. A strong pulpal
inflammation in the carious samples would have been reflected by upregulation of
inflammatory cytokines, e.g. the 17 ILs (IL-1, -1, -2, -4, -5, -6, -7, -8, -9, -10,
-11, -12, -12 -13, -14, -15 and -16) detectable with the cDNA microarray used.
Of these interleukins, only anti-inflammatory IL-13 was strongly expressed.
However, the IL-13 expression was equal in healthy and carious teeth pulp.
Therefore, it can be concluded that the carious pulp samples used in this study
specifically represented pulp cells.
Fig. 5. Schematic figure showing differences between the teeth with small or moderate
caries used in part I of this study and the teeth with deep carious lesions used by
McLachlan et al. (2005)
63
The collection of sufficient numbers of human teeth for in vivo analyses,
especially carious teeth with similar caries in respect to lesion progression and
activity, is difficult and time-consuming. On the other hand, in recent years
several methods, e.g. laser-capture microscopy, enabling the selection of only the
affected portion of sample (Fig. 5.) have emerged. Additionally, microarray
methodology has been developed enabling the use of smaller sample amounts.
Another problem, arising from the samples used in the study, is the variability
between the individual teeth caused by genetic and environmental factors. cDNA
microarray in part I of this study revealed differences between the two individual
samples, while the two hybridizations of the same sample were similar. This
indicated a varying gene expression pattern between the individual teeth. Thus,
the suitability of the large-scale expression screening methods for in vivo pulp
biological studies seems to be poor.
6.1.2 In vitro analysis of the transcriptome and proteome
Culturing of pulp tissues and odontoblasts +/- TGF-1, which is an important

regulator of defense mechanisms of the dentin-pulp complex (Smith 2003),
enabled the study of the potential of large-scale expression analysis in pulp
biology in vitro. Since the samples were handled in strictly regulated conditions,
environmental causes for differences were eliminated. DNA microarray analysis
with oligonucleotide arrays (Affymetrix) revealed notable up- or downregulation
of hundreds of genes both in pulp tissue and in odontoblasts in response to
TGF-1. The more specific effects of TGF-1 on the gene expression of cytokines
are discussed below. Large expression changes, in response to TGF-1 in relation
to the changes caused by caries, were probably caused by the whole extent of the
cultured pulp tissues being affected by TGF-1, while in pulp tissues of carious
teeth only a small amount of tissue was diseased. In recent years, several analyses
of the effects of TGF- on the transcriptome of various tissues and cultured cell
types have been performed using microarray (Ranganathan et al. 2007, Kirwan et
al. 2005, Xie et al. 2003, Kloth et al. 2005). All of these studies demonstrated a
large number of genes with altered expression in response to TGF- treatment. As
expected, however, these studies report different profiles in response to TGF-
treatment in different cells and tissues, since TGF-s have pleiotropic effects on
e.g. proliferation, differentiation, migration and survival of cells, and thus affect
multiple biological processes (Blobe et al. 2000). The effects of TGF-1 on
osteoblast cell line MC3T3-E1 have been studied previously (Kahai et al. 2004),
64
but the expression changes detected were very different from the changes detected
in part III of this study, e.g. in osteoblasts, no increase in IL expression was
reported. Thus, no conclusions concerning dentin-pulp complex cells can be
drawn by extrapolation of results from other tissues to pulp tissue or odontoblasts.
The gene expression changes of a selected gene group, ILs (discussed below in
section 6.5), detected by microarray were successfully confirmed at the protein
level using antibody array. Thus, large-scale screening seems more suitable for in
vitro than in vivo pulp biology research.
6.2 Comparison of microarray with other large-scale transcription

screening methods used in pulp biology
The use of large-scale transcription analysis methods in pulp biology has been
very limited. SSH has been used to screen for odontoblast-specific genes in rat
and human (Buchaille et al. 2000, Dey et al. 2001). These analyses yielded a
considerably scarcer amount of differentially expressed genes than detected by
microarray in this study (II). This was probably due to the differences between the
used methods, even considering that in this study the number of differentially
detected probe sets, not actual transcripts, were calculated. Nonetheless, SSH has
some advantages over microarray. Firstly, it does not require any prior analysis
knowledge of the expression profile that is required when selecting probes for the
microarray. This advantage may no longer be as valid as previously, since several
commercial DNA microarrays include virtually every transcript encoded by the
human genome. The second advantage of SSH is that, unlike the microarray, it
does not require special equipment. On the other hand, performing SSH analysis
requires much more laboratory work in comparison with the microarray, and may
thus be more prone to methodological errors.
6.3 The effect of caries on the expression profile of pulp tissue
Bacterial invasion is the main cause of caries. The response to caries may depend
on microbial flora, caries activity, or the type of tooth (e.g. young or old).
Basically, a local defensive reaction is initiated already in the early stages of
caries with dentin tubule occlusion and formation of reactionary dentin by
primary odontoblasts, accompanied by a localized reaction seen as the
accumulation of pulpal fibroblasts under the odontoblast layer and a low-grade
inflammatory reaction (Brannstrom & Lind 1965, Bjorndal et al. 1998). If the
65
bacterial invasion continues, a more intense defense reaction occurs, observed as
reparative dentin formation by odontoblast-like cells or replacement odontoblasts
and more intense inflammatory reaction (Smith 2003, Trowbridge 2002). If the
bacterial irritation overrides the defensive reaction, widespread inflammation and
necrosis occurs. Therefore, the state of caries may have a significant effect on the
outcome when evaluating the pulpal response, including the large-scale screening
methods used here. The decision to include only teeth with small and medium-
sized caries lesions was based on an attempt to avoid pulp tissue with wide-spread
inflammation; the samples from these teeth better represent the pulp-specific
response, instead of the overall inflammatory response. It should be therefore
realized that the results, at best, represent one phase of the continuum of the
inflammatory pulpal response to caries.
6.3.1 The effect of caries on the transcriptome of pulp tissue
Differential expression of several genes between the pulp tissue of healthy and
carious teeth was detected. In addition, several genes with previously unknown
high expression in pulp tissue were detected. These highly or differentially
expressed genes participate in one or several biological processes that happen in
carious pulp, including growth and differentiation of cells, angiogenesis, nerve
growth and inflammatory response.
Genes with an effect on cell differentiation
During caries, pulp cells are attracted to the injury site and differentiate into
odontoblasts or odontoblast-like cells, in a process involving e.g. Notch signaling
(Mitsiadis & Rahiotis 2004). Two Notch ligands, JAG2 and DLK, were up-
regulated in carious samples (I). The Notch pathway is an evolutionarily
conserved signaling pathway, and mutations in its components disrupt cell fate
specification and embryonic development (Artavanis-Tsakonas et al. 1999). Five
genes encoding ligands for the Notch receptors have been found in mammals:
Jagged 1 and Jagged 2 (JAG1 and -2,) and Delta-like 1 and Delta-like 3
(DLL1, -3 and -4). JAG2 and DLL are involved in cell fate specification of
tissues, which depends on epithelial-mesenchymal interactions, including
odontogenesis (Valsecchi et al. 1997, Mitsiadis et al. 1998). Notch signaling is
typically restricted to prenatal tissues; however, reactivation of Notch expression
has been detected in rat pulp after experimental pulp exposure (Mitsiadis et al.
66
1999). The activation of the Notch pathway is thought to ensure the supply for
progenitor cells capable of differentiation into replacement odontoblasts
(Mitsiadis et al. 1999, Mitsiadis et al. 2003). Notch signaling through JAG1-
ligand has been shown to inhibit odontoblast differentiation (Zhang et al.
2008).The detection of upregulation of Notch ligands JAG2 and DLK in carious
pulp in part I of this study confirms the previous findings of induced Notch
signaling under caries lesions and experimental cavity preparations (Mitsiadis et
al. 1999, Mitsiadis et al. 2003, Lovschall et al. 2005).
Placental growth factor
A high PlGF expression level was detected both in healthy and carious teeth pulp
with upregulation in response to caries (I). PlGF is a member of the vascular
endothelial growth factor (VEGF) family and significantly potentiates VEGF
function (Park et al. 1994). Pulp fibroblasts have recently been indicated to play
an important role in pulp angiogenesis by secreting VEGF and fibroblast growth
factor 2 (FGF-2) (Tran-Hung et al. 2006). Interestingly, a small VEGF
downregulation in teeth with irreversible pulpitis has been observed (Artese et al.
2002), while in part I of this study the great upregulation of PlGF gene expression
was observed in carious pulp samples. Thus, PlGF may be an important
regulatory factor in both healthy and carious teeth pulp tissue.
Matrix metalloproteinase 13
High MMP-13 gene expression was detected in the pulp tissue of both healthy
and carious teeth by cDNA microarray (I). MMP-13 degrades all fibrillar
collagens, but is most potent against type II collagen, and also several other ECM
molecules, including types IV, IX, X, and XIV collagens; fibronectin; and
tenascin-C (Knauper et al. 1997). Overall in normal adult tissues, the expression
of MMP-13 is low or non-existent (Freije et al. 1994, Brinckerhoff et al. 2000,
Leeman et al. 2002). Thus, high and equal expression in healthy and carious teeth
pulp tissue suggests that MMP-13 has an important role in ECM turnover in pulp
tissue.
67
6.3.2 The effect of caries on the proteome of pulp tissue
2-D gel electrophoresis and MS revealed the identities of 96 proteins in the

healthy pulp tissue, but no consistent differences between healthy and diseased
pulp tissues could be detected. Searching the SWISS-PROT
(http://www.ebi.ac.uk/swissprot/) database revealed that the majority of proteins
encoded by these genes are secreted (e.g. gene products of OSM, ET-2, GDF-1,
TNF-R1, NGF-2, IL-13, MMP-13). Due to the limited protein amount, the 2-D
analyses focused mainly on the whole proteome of the pulp tissue. Therefore,
only a limited number of proteins from the different intra- and extracellular
fractions (e.g. secreted proteins) might be detectable in the 2-D gel. On the other
hand, several of these proteins are integral membrane proteins (PlGF, EDG4,
DARC) with known low solubility. The ability of 2-D gel electrophoresis to
separate hydrophobic membrane proteins is limited, which may explain the
missing identification of these gene products. Therefore, it is possible that the
differential mRNA expression detected using cDNA microarray is also reflected
at the protein level.
Novel protein detected in pulp tissue
The 2-D electrophoresis revealed the presence of putative nucleoside diphosphate

(NDP) kinase (H_278C19.3), whose function in cells is unknown. The NDP
kinases are a large family of structurally and functionally conserved proteins that
generally catalyze the transfer of -phosphates between nucleoside tri- and
diphosphates (Lascu & Gonin 2000). There is also evidence suggesting that in
addition to their basic enzymatic activity, NDP kinases might have other functions
related to signal transduction, cell growth and differentiation, embryonic
development, tumor progression, metastasis and apoptosis (Hartsough & Steeg
2000, Kimura et al. 2000, Lacombe et al. 2000). The exact location and the role
of putative NDP kinase (H_278C19.3) in pulp tissue remain unclear and would
require further analysis.

tissue and odontoblasts
Various growth factors, including IGF-I and -II, BMPs and several angiogenic
growth factors (Finkelman et al. 1990, Roberts-Clark & Smith 2000, Bessho et al.
68
1991), have been detected in dentin, but TGF-1 is the most extensively studied
growth factor in the dentinpulp complex. It has been indicated in the regulation
of tooth development and dentin ECM production (Begue-Kirn et al. 1992, Sloan
& Smith 1999). TGF- isoforms 1, 2 and 3 are produced by odontoblasts of
mature human teeth (Sloan et al. 2000) and become sequestered in the dentin
(Finkelman et al. 1990, Cassidy et al. 1997). TGF- has been shown to stimulate
odontoblast differentiation and reparative dentinogenesis. It has also been
speculated to function in the regulation of pulp inflammatory reactions (Smith
2003). Upon dentinal damage (such as caries), dentin-bound TGF-1 is believed
to be released and to act as a stimulatory factor for odontoblast-like cell
differentiation and extracellular matrix synthesis, resulting in reparative or
reactionary dentin formation (Tziafas et al. 2000). The effects of TGF- on cells
are largely carried out by the phosphorylation of mothers against decapentaplegic
homolog 2/3 (SMAD 2/3) by the activated TGF- type I receptor (TGFBRI) (Shi
& Massague 2003). Several TGF--related genes, including TGFBRI, SMAD2,
SMAD4 and ectopic viral integration site 1 (EVI1), are expressed in odontoblasts
(Sloan et al. 2000, Lucchini et al. 2002, McLachlan et al. 2003, Durand et al.
2007, Hwang et al. 2008).
TGF-1 treatment caused considerable changes in the gene expression
profiles of both pulp tissue and odontoblasts. Very high, over 8-fold, changes
were detected in 127 genes in pulp and 122 in odontoblasts. In addition, mere
culturing itself caused notable expression changes, probably reflecting
environmental changes caused by extraction of the tissues and the presence of
growth factors and other substances in the culture media. In the samples cultured
for 1 h and 5 h, representing the early response to TGF-1 exposure, several
cytokine-related GO categories (e.g. chemokine activity, chemotaxis, cytokine
activity and inflammatory response) were among the most significantly altered
categories. This indicates that TGF-1 is, along with or instead of bacterial
products, an important regulator of at least the initial pulpal inflammation. This is
supported by the demonstration of localized inflammatory reaction under shallow
carious lesions (Bergenholtz 2000) and the proinflammatory effect of TGF-1 in
in vitro experiments using the dentin-pulp complex slice culturing method (Farges
et al. 2003). Interestingly, analysis of odontoblast samples did not reveal any GO
categories with significant changes in response to TGF-1 treatment at both the 1
h and 24 h time points. This is probably due to the long time period between the
culturing times, 1 h and 24 h. Odontoblast response to short and long-term
69
culturing may be differential. Since at least five samples are required for reliable
dChip analysis, independent analysis of culture times was impossible.
6.5 Cytokine profile of pulp tissue and odontoblasts
The expression of many inflammation and immune system related genes is

elevated in pulp tissue during caries (Zehnder et al. 2003, McLachlan et al. 2004).
TGF-1 induces the accumulation of dendritic cells into the odontoblast layer
indicating that TGF-1 plays a role in the immune response of dental pulp (Farges
et al. 2003). Because of the important and complex role of cytokines in the
regulation of healthy tissue and inflammatory reactions, they were selected for
detailed analysis as a model for the large-scale gene and protein expression
analysis in pulp biological experiments. Several genes encoding inflammatory
cytokines demonstrated interesting expression patterns; in particular the
expression of several ILs was transiently elevated after TGF-1 treatment of pulp
tissue.
6.5.1 Interleukin profile of pulp tissue
In part III of this study, the gene expression of three proinflammatory cytokines,
IL-7, -12 and -16, was detected in native pulp tissue. IL-7 is known to act as a
growth factor for B- and T-lymphocytes (Feghali & Wright 1997), while IL-12
has been shown to enhance cytotoxic T cells as well as increase NK cell
proliferation (Feghali & Wright 1997), and IL-16 is a chemoattractant for T cells,
eosinophils, monocytes and dendritic cells (Cruikshank & Center 1982, Rand et
al. 1991, Kaser et al. 1999, Cruikshank et al. 1987). The mRNA of anti-
inflammatory IL-13 was detected both in native intact and carious pulp tissue in
part I of this study. However, in part III it was not observed either in native intact
or in +/- TGF-1 cultured pulp tissue (detection p-value over 0.1 in each sample).
This inconsistency between the results is possibly due to different probe
sequences on the arrays used. Also, since the result was not confirmed with any
other method, it is possible that it represents a false positive finding.
After TGF-1 treatment, proinflammatory cytokines IL-1, -1, -6, -8,
-11, -16 and -23 all were transiently elevated, at least 2-fold, at the mRNA level,
and IL-1, -1 and -6 also at the protein level. However, the increase in IL-8
expression observed at the mRNA level was not detected at the protein level,
possibly indicating downregulation of IL-8 at the protein level or downregulation
70
of IL-8 secretion. The differential cytokine response, at the mRNA and protein
levels, to treatment with other cytokines has been reported previously, e.g.
induction of TGF- protein with no change in mRNA amounts in IL-2-treated
macrophages (Nelson et al. 1994). Induced IL-8 expression in cultured pulp cells
in response to nitric oxide treatment has been reported (Min et al. 2008). The
much higher IL-6 and -8 amounts compared with the levels of other ILs included
on the antibody array most likely indicated the importance of IL-6 and -8 in
triggering the immune response of pulp tissue.
All these ILs play important roles in chemotaxis and lymphocyte stimulation
(Feghali & Wright 1997, Oppmann et al. 2000, Cruikshank et al. 1994). Lack of
IL-6 has been shown to enhance periapical lesion development, thus indicating
the importance of IL-6 in pulp immune defense (Huang et al. 2001). IL-6
expression by human pulp fibroblasts is induced by proinflammatory cytokines
IL-1 and TNF- (Yang et al. 2003b, Lin et al. 2002) suggesting that these
proinflammatory cytokines are involved in the pathogenesis of pulpitis. IL-2, a
growth factor/activator for T cells, NK cells and B cells, has earlier been observed
both in healthy and inflamed pulp tissue (Anderson et al. 2002, Bletsa et al. 2006),
but in parts I and III of this study no IL-2 mRNA was detected using microarray
analysis. After TGF-1 treatment, elevated amounts of proinflammatory IL-2, -7
and -12 were detected at the protein level but not at the mRNA level, possibly
reflecting a low mRNA expression level of IL-2 (detection p-values over 0.2)
falling under the detection level of the microarray used, and upregulation of IL-7
and -12 at the protein level.
Anti-inflammatory IL-4 and -10 have been earlier shown to be present in
inflamed pulp tissue (Hahn et al. 2000), but in this study (I, III) neither of them
was detected at the mRNA level, nor were any changes in their protein expression
observed after TGF-1 treatment. Since TGF-1 treatment clearly induced the
gene and protein expression of the proinflammatory cytokines, but caused no
changes in the level of anti-inflammatory cytokines, it can be concluded that
TGF-1 had predominantly proinflammatory effects on the dentin-pulp complex.
TGF-1 is known to be proinflammatory during the initial stages of pulpal
inflammation and to function in recruiting immunocompetent cells (Farges et al.
2003). At the later stages of inflammation, TGF-1 has anti-inflammatory effects
through the suppression of lymphocyte proliferation and the activation of
dendritic cells and macrophages (DSouza et al. 1998). The tissue culture periods
used (maximum of 48 h) may have been too short for the anti-inflammatory
71
effects of TGF-1 to appear. Another possibility is that inflammatory cells, which
are not present in the tissue cultures, mediate these effects in vivo.
6.5.2 Interleukin profile of odontoblasts
A smaller number of interleukins was detected in odontoblasts than in pulp tissue,

probably since the odontoblast layer consists of one cell type while pulp tissue
contains various different cell types, or since odontoblasts are highly specialized
cells. Most cell types are able to produce various cytokines when simulated by
diverse factors (Oppenheim & Feldmann 2003).
In native odontoblasts, the gene expression of only two ILs, IL-6 and -7, both
proinflammatory cytokines with functions in growth and stimulation of
lymphocytes (Feghali & Wright 1997), was detected. While IL-7 could be directly
amplified by RT-PCR from native pulp tissue, nested-PCR was required for its
detection from odontoblasts. Thus, the IL-7 gene expression level in odontoblasts
is probably lower than in pulp tissue. In addition to its proinflammatory function,
IL-7 inhibits osteoblast differentiation, thereby reducing bone formation (Giuliani
et al. 2005). Therefore, it may play a role also in the regulation of either
reactionary or reparative dentin formation, or both. IL-6 gene expression has not
been detected in odontoblasts previously. IL-6 is a growth factor for B-
lymphocytes and plays a role in their differentiation and maturation into plasma
cells. Additionally, IL-6 plays a role in the activation of T-lymphocytes (Feghali
& Wright 1997). Chemokines that are constitutively expressed by non-lymphoid
tissues, such as skin and salivary glands, are important in immunosurveillance
and homeostasis of the whole immune system (Mantovani et al. 2004). Durand et
al. (2006) demonstrated that odontoblasts obtained from normal pulp
constitutively expressed 17 chemokine-related genes, among them CCL2,
CXCL12 and CXCL14, known to recruit immature dendritic cells and
macrophages. ILs now detected in odontoblasts may play similar roles in the
immunosurveillance and homeostasis of teeth.
The expression of only one IL, proinflammatory IL-8, was clearly elevated in
response to TGF-1 treatment of odontoblasts. IL-8 could not be detected in
native odontoblasts, supporting the earlier evidence of non-existent or very weak
IL-8 gene expression in native odontoblasts (Veerayutthwilai et al. 2007, Levin et
al. 1999). An increase in the expression levels of IL-8 in odontoblasts in response
to culture and treatment with lipopolysaccharides (LPS) has been reported
(Veerayutthwilai et al. 2007, Levin et al. 1999). Thus, the findings of this study
72
(III) support the evidence that odontoblast expression of IL-8 is upregulated by
extrinsic and intrinsic factors, and IL-8 may be a key cytokine in the odontoblast-
mediated immune response.
6.6 Potential novel odontoblast markers
The DSPP gene encodes both dentin sialoprotein (DSP) and dentin
phosphoprotein (DPP), two principal dentin-specific noncollagenous proteins (Gu
et al. 2000). DSPP expression has traditionally been thought to be odontoblast-
specific. However, DSPP mRNA and the corresponding DSP protein have also
been detected at lower levels in e.g. alveolar bone, cellular cementum and
periodontium (Baba et al. 2004). Additionally, they have been detected at low
levels in non-dental tissues, including murine long bone and mouse inner ear (Qin
et al. 2002, Xiao et al. 2001). In part II of this study, DSPP mRNA was detected
in both native and cultured odontoblasts, but also at a lower amount in each of the
pulp samples, with no marked effect of TGF-1. The detection of DSPP mRNA in
pulp tissue is consistent with the earlier observation that DSP encoded by the
DSPP gene is present in rat pulp tissue (Baba et al. 2004). Thus, DSPP is not
really odontoblast-specific, even in dental tissues, and cannot be used as a sole
marker in odontoblast differentiation studies or other experiments with cultured
odontoblast-like cells. The expression of other proteins, including osteoadherin,
type I collagen, enamelysin and alkaline phosphatase (Couble et al. 2000, Allard
et al. 2000, Kitagawa et al. 2007), have also been traditionally used in
determining the differentiation stage of odontoblast-like cells. None of these
proteins is odontoblast-specific, and a combination of different markers must be
used to conclude that given a cell has odontoblast characteristics. Novel protein
HUGO (FNDC3A) has been recently identified from cultured odontoblast-like
cells (Carrouel et al. 2008). However, it is expressed also in other tissues such as
brain and kidney (Carrouel et al. 2008). Thus, the discovery of novel odontoblast-
specific genes would benefit studies of odontoblast differentiation.
6.6.1 Matrilin 4 and collagen XI 1
Comparison of native pulp tissue and odontoblasts (II) revealed that, despite the
functional diversity of the tissues and the composition of pulp tissue of several
cell types, the general expression profiles were surprisingly similar. Since
odontoblasts are hard tissue-forming cells responsible for dentin formation, major
73
differences especially in the expression of ECM organization and biogenesis-
related genes were expected. However, only two genes of this category,
COL11A1 and MATN4, were detected solely in odontoblasts with microarray
analysis. At the closer analysis with RT-PCR, COL11A1 was also observed in
pulp tissue, while MATN4 mRNA and protein were confirmed only in
odontoblasts. The expression profiles of two healthy pulp samples downloaded
from the GEO database (GSE1629) revealed COL11A1 expression in these pulp
samples. This may be due to the biological variation between teeth (caused by e.g.
genetic and environmental factors) used for studies and the slight variation in
microarray methodology. No MATN4 expression was detected in either of the two
samples downloaded from the GEO database. However, MATN4 is expressed by
several tissues including bone and cartilage in rat (Klatt et al. 2001), and it is also
possible that cells of these lineages, differentiated from pulp stem cells, may
express it. Thus, MATN4 could be used as an odontoblast differentiation marker
in combination with other markers. The specific function of matrilins is unclear,
but they have been shown to interact with both collagenous and noncollagenous
matrix proteins (Piecha et al. 2002, Wiberg et al. 2003) suggesting a function as
general adaptor proteins in the ECM.
6.6.2 The expressed sequence tags detected only in odontoblasts
Microarray analysis revealed 16 probe sets representing ESTs and other

unclassified transcripts, which were positive in native odontoblasts but not in
native pulp tissue or in any of the cultured pulp samples. Two of them were
consistently expressed in all cultured odontoblast samples, and thus possibly
important for odontoblast function. The genes represented by the observed ESTs
and proteins they encode are previously unknown. Since to date their expression
has not been detected in any tissues, they may be odontoblast-specific and may
have a distinct role in the normal function of odontoblasts. The expression of
these ESTs in other tissues requires further analysis, but since they are
odontoblast-specific in tooth, some of them may be good candidates for
odontoblast markers.
74
6.7 Similarity of cultured odontoblast-like cells to mature native
odontoblasts
Dental pulp stem cells are thought to be derived from perivascular cells of pulp
tissue (Shi & Gronthos 2003). Stem cells capable of differentiating into various
cell types and generating dentin-pulp complex -like structures, when transplanted
into mouse, have been isolated from human pulp tissue (Gronthos et al. 2002,
Gronthos et al. 2000). Differentiation of odontoblast-like cell lines from dental
papilla or pulp tissue can be induced by -glyserophosphate (GP) or isolated
dentin matrix components (Couble et al. 2000, Liu et al. 2007, About et al. 2000,
Liu et al. 2005). These cells typically possess some odontoblast-like
characteristics, e.g. expression of DSP protein, and are capable of producing small
mineralized nodules (Liu et al. 2007, About et al. 2000). Additionally, to
circumvent the limited life-span of primary cultures, immortalized cell lines
representing odontoblast-like cells have been established by reconstitution of
telomerase activity (Hao et al. 2002) or retroviral transcription (MacDougall et al.
1995), or even spontaneous immortalization of pulp cells (Hanks et al. 1998).
These immortalized cell lines may express DSP, alkaline phosphatase or other
odontoblast-characteristic proteins, but their morphology differs greatly from
odontoblasts, e.g. they may have multiple short cell processes (MacDougall et al.
1995, Hanks et al. 1998). The cells immortalized by telomerase activity have a
slightly elongated shape, express some genes that are characteristic for
odontoblasts, and also have the ability to form small mineralized nodules (Hao et
al. 2002). However, since the observed phenotype and spatial organization of
these immortalized cells differ from the native cells, and native odontoblasts are
non-proliferating terminally differentiated cells, it is questionable that these
immortal cell lines truly represent real odontoblasts.
The cell culture system used in part IV of this study allows the differentiation
of human pulp cells into odontoblast-like cells that have the morphological
(position of the nucleus, cellular extension), spatial organization (arrangement
into single-cell layers) (Couble et al. 2000) and the functional (expression of
DSPP, type I collagen, enamelysin, osteoadherin) characteristics of odontoblasts
(Couble et al. 2000, Allard et al. 2000). The marked similarity of general gene
expression profiles of the mature native odontoblasts and cultured odontoblast-
like cells was revealed by microarray analysis. Distribution of the positive probe
sets into GO categories was surprisingly similar considering that the samples
represented mature and newly differentiated cells. However, differences were also
75
observed, especially cell growth and cell cycle categories, which held more
positive probe sets in cultured than in native odontoblasts. The difference was
probably partly explained by the culture conditions, but also by the newly
differentiated status of the cultured cells and terminally differentiated stage of the
native odontoblasts. The detection of stimulus was the only category with more
positive probe sets in native odontoblasts than in cultured odontoblast-like cells,
possibly due to the suggested function of odontoblasts as sensory cells (Magloire
et al. 2004). Thus, the large-scale gene expression screening confirmed the earlier
findings (Couble et al. 2000, Allard et al. 2000) that the cells produced by this
culture method have the characteristics of odontoblasts.
6.7.1 Neuronal protein expression in mature and newly differentiated

odontoblasts
In addition to their established function in dentin matrix production and

mineralization, odontoblasts have been indicated to participate in the sensory
function of tooth. The evidence for this is still limited, although mechanosensitive
potassium channels (Allard et al. 2000, Magloire et al. 2003) and voltage-gated
calcium channels (Lundgren & Linde 1997, Davidson & Guo 2000) have been
detected in the odontoblast plasma membrane. Neuropeptide NT has been
detected in odontoblasts earlier and has been speculated to participate in the
nociceptive function of tooth (Bhatnagar et al. 1995). Adhesion-like contacts have
been observed between odontoblasts and nerve fibers, but there is no evidence of
synapses between them (Byers 1984, Byers & Nrhi 1999). However, due to their
close spatial location, communication between odontoblasts and nerve fibers is
probable, e.g. by paracrine factors. Thus, the expression of selected neuronal
genes, including SSTR1, CORT, PNOC GAL and PTPRZ1, in mature native and
in newly differentiated odontoblasts was selected for detailed analysis. At the
mRNA level, higher CORT, PTPRZ1, GAL and SSTR expression was detected in
native than in cultured odontoblasts, while the PNOC level was higher in cultured
odontoblasts. None of these neuronal proteins have been previously reported in
odontoblasts. However, their precise roles in odontoblasts remain to be studied at
the functional level.
Immunofluorescent analysis was performed only on developing teeth, which
nonetheless contained both mature (at the crown) and newly differentiated (at the
apical part) odontoblasts. None of the studied proteins were detected in pulp cells.
A similar expression pattern as with DNA microarray and real-time qPCR was
76
detected, confirming the strength of the DNA microarray as a tool for screening
previously unknown and potentially functionally important proteins in cells of the
dentin-pulp complex.
Since PNOC was detected at a higher level in newly differentiated than in
mature odontoblasts, it may be useful as an odontoblast marker. PNOC is a
precursor of nociceptin that has a nociceptive function in cells (Mollereau et al.
1996). Further investigation at the functional level is required to elucidate the role
of PNOC in odontoblasts.
77
78
Conclusions
In the present study, the suitability of high-throughput expression analysis
methods for pulp biology was estimated, and the expression profiles of native and
cultured pulp tissue as well as odontoblasts and odontoblast-like cells were
investigated.
1. Only slight differences between healthy and carious pulp tissue were seen in
vivo at the mRNA level, and no reproducible differences were observed at the
protein level. On the other hand, treatment of pulp tissue with TGF-1 in
vitro resulted in large changes, which were confirmed at the protein level.
Thus, large-scale expression analysis is especially suitable for in vitro studies
in pulp biology.
2. Comparison of a native pulp tissue sample used in this study with two
samples downloaded from the GEO database confirmed the good
reproducibility of the Affymetrix GeneChip array, which has been previously
shown (Lockhart et al. 1996), also in pulp tissue.
3. TGF-1 treatment caused marked changes in the transcriptome of pulp tissue
and odontoblasts. Cytokines were one of the most significantly altered gene
categories in pulp tissue. Especially, the expression of proinflammatory
interleukins was transiently increased in pulp tissue in response to TGF-1,
indicating the predominantly proinflammatory effect of TGF-1 on pulp
tissue. IL-8 was the only interleukin with an observed gene expression
change in odontoblasts, and thus it may be the key cytokine in the immune
response mediated by odontoblasts.
4. The previous findings of dentin sialophosphoprotein (DSPP) expression in
pulp tissue were confirmed, thus emphasizing the demand for new
odontoblast markers. Matrilin 4 (MATN4) was identified as odontoblast-
specific, at the mRNA and protein levels, in tooth. Additionally, two
potentially odontoblast-specific ESTs, representing possible new odontoblast-
specific genes, were identified.
5. The large-scale gene expression screening confirmed the earlier findings that
the cells produced by the culture method (Couble et al. 2000) used in this
study have odontoblast characteristics, and thus offer a valuable cell line for
in vitro studies of odontoblasts. Also, differential expression levels of selected
neuronal proteins were detected in mature native odontoblasts and cultured
odontoblast-like cells, and these proteins, especially pronociceptin (PNOC)
79
detected only in newly differentiated odontoblasts, may also prove useful as
odontoblast markers.
80
References
Aatsinki JT (1997) Coupled one-step reverse transcription and polymerase chain reaction
procedure for cloning large cDNA fragments. Methods Mol Biol 67: 5560.
Abdueva D, Wing MR, Schaub B & Triche TJ (2007) Experimental comparison and
evaluation of the Affymetrix exon and U133Plus2 GeneChip arrays. PLoS ONE 2(9):
e913.
About I, Bottero MJ, de Denato P, Camps J, Franquin JC & Mitsiadis TA (2000) Human
dentin production in vitro. Exp Cell Res 258(1): 3341.
Allard B, Couble ML, Magloire H & Bleicher F (2000) Characterization and gene
expression of high conductance calcium-activated potassium channels displaying
mechanosensitivity in human odontoblasts. J Biol Chem 275(33): 2555625561.
Anderson L & Seilhamer J (1997) A comparison of selected mRNA and protein
abundances in human liver. Electrophoresis 18(34): 533537.
Anderson LM, Dumsha TC, McDonald NJ & Spitznagel JK Jr (2002) Evaluating IL-2
levels in human pulp tissue. J Endod 28(9): 651655.
Arana-Chavez VE & Massa LF (2004) Odontoblasts: the cells forming and maintaining
dentine. Int J Biochem Cell Biol 36(8): 13671373.
Artavanis-Tsakonas S, Rand MD & Lake RJ (1999) Notch signaling: cell fate control and
signal integration in development. Science 284(5415): 770776.
Artese L, Rubini C, Ferrero G, Fioroni M, Santinelli A & Piattelli A (2002) Vascular
endothelial growth factor (VEGF) expression in healthy and inflamed human dental
pulps. J Endod 28(1): 2023.
Baba O, Qin C, Brunn JC, Jones JE, Wygant JN, McIntyre BW & Butler WT (2004)
Detection of dentin sialoprotein in rat periodontium. Eur J Oral Sci 112(2): 163170.
Barreda DR, Hanington PC & Belosevic M (2004) Regulation of myeloid development
and function by colony stimulating factors. Dev Comp Immunol 28(5): 509554.
Begue-Kirn C, Smith AJ, Ruch JV, Wozney JM, Purchio A, Hartmann D & Lesot H (1992)
Effects of dentin proteins, transforming growth factor beta 1 (TGF beta 1) and bone
morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro. Int J
Dev Biol 36(4): 491503.
Bergenholtz G (2000) Evidence for bacterial causation of adverse pulpal responses in
resin-based dental restorations. Crit Rev Oral Biol Med 11(4): 467480.
Berggreen E & Heyeraas KJ (2000) Effect of the sensory neuropeptide antagonists h-
CGRP((837)) and SR 140.33 on pulpal and gingival blood flow in ferrets. Arch Oral
Biol 45(7): 537542.
Bertone P & Snyder M (2005) Advances in functional protein microarray technology.
FEBS J 272(21): 54005411.
Bessho K, Tanaka N, Matsumoto J, Tagawa T & Murata M (1991) Human dentin-matrix-
derived bone morphogenetic protein. J Dent Res 70(3): 171175.
Bhatnagar M, Cintra A, Tinner B, Agnati LF, Kerezoudis N, Edwall L & Fuxe K (1995)
Neurotensin-like immunoreactivity in odontoblasts and their processes in rat maxillary
molar teeth and the effect of pulpotomy. Regul Pept 58(3): 141147.
81
Bjorndal L, Darvann T & Thylstrup A (1998) A quantitative light microscopic study of the
odontoblast and subodontoblastic reactions to active and arrested enamel caries
without cavitation. Caries Res 32(1): 5969.
Bjorndal L & Mjor IA (2001) Pulp-dentin biology in restorative dentistry. Part 4: Dental
caries--characteristics of lesions and pulpal reactions. Quintessence Int 32(9): 717
736.
Bletsa A, Berggreen E, Fristad I, Tenstad O & Wiig H (2006) Cytokine signalling in rat
pulp interstitial fluid and transcapillary fluid exchange during lipopolysaccharide-
induced acute inflammation. J Physiol 573(Pt 1): 225236.
Bletsa A, Heyeraas KJ, Haug SR & Berggreen E (2004) IL-1 alpha and TNF-alpha
expression in rat periapical lesions and dental pulp after unilateral sympathectomy.
Neuroimmunomodulation 11(6): 376384.
Blobe GC, Schiemann WP & Lodish HF (2000) Role of transforming growth factor beta in
human disease. N Engl J Med 342(18): 13501358.
Blum H, Beier H & Gross HJ (1987) Improved silver staining of plant proteins, RNA and
DNA in polyacrylamide gels. Electrophoresis 8(2): 9399.
Bongenhielm U, Haegerstrand A, Theodorsson E & Fried K (1995) Effects of
neuropeptides on growth of cultivated rat molar pulp fibroblasts. Regul Pept 60(23):
9198.
Brannstrom M & Lind PO (1965) Pulpal response to early dental caries. J Dent Res 44(5):
10451050.
Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, Aach J,
Ansorge W, Ball CA, Causton HC, Gaasterland T, Glenisson P, Holstege FC, Kim IF,
Markowitz V, Matese JC, Parkinson H, Robinson A, Sarkans U, Schulze-Kremer S,
Stewart J, Taylor R, Vilo J & Vingron M (2001) Minimum information about a
microarray experiment (MIAME)-toward standards for microarray data. Nat Genet
29(4): 365371.
Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S,
Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR,
Moon K, Burcham T, Pallas M, DuBridge RB, Kirchner J, Fearon K, Mao J &
Corcoran K (2000) Gene expression analysis by massively parallel signature
sequencing (MPSS) on microbead arrays. Nat Biotechnol 18(6): 630634.
Brinckerhoff CE, Rutter JL & Benbow U (2000) Interstitial collagenases as markers of
tumor progression. Clin Cancer Res 6(12): 48234830.
Buchaille R, Couble ML, Magloire H & Bleicher F (2000) A substractive PCR-based
cDNA library from human odontoblast cells: identification of novel genes expressed
in tooth forming cells. Matrix Biol 19(5): 421430.
Butler WT, Brunn JC & Qin C (2003) Dentin extracellular matrix (ECM) proteins:
comparison to bone ECM and contribution to dynamics of dentinogenesis. Connect
Tissue Res 44 Suppl 1: 171178.
Buttner K, Bernhardt J, Scharf C, Schmid R, Mader U, Eymann C, Antelmann H, Volker
A, Volker U & Hecker M (2001) A comprehensive two-dimensional map of cytosolic
proteins of Bacillus subtilis. Electrophoresis 22(14): 29082935.
82
Byers MR (1984) Dental sensory receptors. Int Rev Neurobiol 25: 3994.
Byers MR & Nrhi MV (1999) Dental injury models: experimental tools for understanding
neuroinflammatory interactions and polymodal nociceptor functions. Crit Rev Oral
Biol Med 10(1):439.
Byers MR, Suzuki H & Maeda T (2003) Dental neuroplasticity, neuro-pulpal interactions,
and nerve regeneration. Microsc Res Tech 60(5):503515.
Canales RD, Luo Y, Willey JC, Austermiller B, Barbacioru CC, Boysen C, Hunkapiller K,
Jensen RV, Knight CR, Lee KY, Ma Y, Maqsodi B, Papallo A, Peters EH, Poulter K,
Ruppel PL, Samaha RR, Shi L, Yang W, Zhang L & Goodsaid FM (2006) Evaluation
of DNA microarray results with quantitative gene expression platforms. Nat
Biotechnol 24(9): 11151122.
Carrouel F, Couble ML, Vanbelle C, Staquet MJ, Magloire H & Bleicher F (2008) HUGO
(FNDC3A): a new gene overexpressed in human odontoblasts. J Dent Res 87(2): 131
136.
Cassidy N, Fahey M, Prime SS & Smith AJ (1997) Comparative analysis of transforming
growth factor-beta isoforms 13 in human and rabbit dentine matrices. Arch Oral Biol
42(3): 219223.
Chen S, Santos L, Wu Y, Vuong R, Gay I, Schulze J, Chuang HH & MacDougall M (2005)
Altered gene expression in human cleidocranial dysplasia dental pulp cells. Arch Oral
Biol 50(2): 227236.
Coil J, Tam E & Waterfield JD (2004) Proinflammatory cytokine profiles in pulp
fibroblasts stimulated with lipopolysaccharide and methyl mercaptan. J Endod 30(2):
8891.
Couble ML, Farges JC, Bleicher F, Perrat-Mabillon B, Boudeulle M & Magloire H (2000)
Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif
Tissue Int 66(2): 129138.
Cruikshank W & Center DM (1982) Modulation of lymphocyte migration by human
lymphokines. II. Purification of a lymphotactic factor (LCF). J Immunol 128(6):
25692574.
Cruikshank WW, Berman JS, Theodore AC, Bernardo J & Center DM (1987) Lymphokine
activation of T4+ T lymphocytes and monocytes. J Immunol 138(11): 38173823.
Cruikshank WW, Center DM, Nisar N, Wu M, Natke B, Theodore AC & Kornfeld H
(1994) Molecular and functional analysis of a lymphocyte chemoattractant factor:
association of biologic function with CD4 expression. Proc Natl Acad Sci U.S.A.
91(11): 51095113.
Davidson RM & Guo L (2000) Calcium channel current in rat dental pulp cells. J Membr
Biol 178(1): 2130.
Dey R, Son HH & Cho MI (2001) Isolation and partial sequencing of potentially
odontoblast-specific/enriched rat cDNA clones obtained by suppression subtractive
hybridization. Arch Oral Biol 46(3): 249260.
83
Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S,
Lukyanov K, Gurskaya N, Sverdlov ED & Siebert PD (1996) Suppression subtractive
hybridization: a method for generating differentially regulated or tissue-specific
cDNA probes and libraries. Proc Natl Acad Sci U.S.A. 93(12): 60256030.
Dommisch H, Winter J, Acil Y, Dunsche A, Tiemann M & Jepsen S (2005) Human beta-
defensin (hBD-1, -2) expression in dental pulp. Oral Microbiol Immunol 20(3): 163
166.
DSouza R (2002) Development of the PulpoDentin Complex. In: Hargreaves KM &
Goodis HE (eds) Selzer and Benders Dental Pulp. 3 Illinois: Quintessence Publishing
Co, Inc, 1340.
DSouza RN, Cavender A, Dickinson D, Roberts A & Letterio J (1998) TGF-beta1 is
essential for the homeostasis of the dentin-pulp complex. Eur J Oral Sci 106 Suppl 1:
185191.
Durand SH, Flacher V, Romeas A, Carrouel F, Colomb E, Vincent C, Magloire H, Couble
ML, Bleicher F, Staquet MJ, Lebecque S & Farges JC (2006) Lipoteichoic acid
increases TLR and functional chemokine expression while reducing dentin formation
in in vitro differentiated human odontoblasts. J Immunol 176(5): 28802887.
Durand SH, Romeas A, Couble ML, Langlois D, Li JY, Magloire H, Bleicher F, Staquet
MJ & Farges JC (2007) Expression of the TGF-beta/BMP inhibitor EVI1 in human
dental pulp cells. Arch Oral Biol 52(8): 712719.
Farges JC, Romeas A, Melin M, Pin JJ, Lebecque S, Lucchini M, Bleicher F & Magloire H
(2003) TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer. J
Dent Res 82(8): 652656.
Feghali CA & Wright TM (1997) Cytokines in acute and chronic inflammation. Front
Biosci 2: d1226.
Finkelman RD, Mohan S, Jennings JC, Taylor AK, Jepsen S & Baylink DJ (1990)
Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin. J
Bone Miner Res 5(7): 717723.
Fouad AF (2002) Molecular Mediators of Pulpal Inflammation. In: Anonymous Seltzer
and Benders Dental Pulp. 3 Illinois: Quintessence Publishing Co, Inc, 247279.
Freije JM, Diez-Itza I, Balbin M, Sanchez LM, Blasco R, Tolivia J & Lopez-Otin C (1994)
Molecular cloning and expression of collagenase-3, a novel human matrix
metalloproteinase produced by breast carcinomas. J Biol Chem 269(24): 16766
16773.
Fristad I, Kvinnsland IH, Jonsson R & Heyeraas KJ (1997) Effect of intermittent long-
lasting electrical tooth stimulation on pulpal blood flow and immunocompetent cells:
a hemodynamic and immunohistochemical study in young rat molars. Exp Neurol
146(1): 230239.
Fristad I, Vandevska-Radunovic V, Fjeld K, Wimalawansa SJ & Hals Kvinnsland I (2003)
NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues, and in bone
and dental hard tissue cells. Cell Tissue Res 311(3): 383391.
Garant PR (2003a) Dentin. In: Garant PR (ed) Oral Cells and Tissues. Illinois:
Quintessence Publishing Co, Inc, 2552.
84
Garant PR (2003b) Early Tooth Development. In: Garant PR (ed) Oral Cell and Tissues.
Illinois: Quintessence Co, Inc., 124.
Ginsburg I (2002) Role of lipoteichoic acid in infection and inflammation. Lancet Infect
Dis 2(3): 171179.
Giuliani N, Colla S, Morandi F, Lazzaretti M, Sala R, Bonomini S, Grano M, Colucci S,
Svaldi M & Rizzoli V (2005) Myeloma cells block RUNX2/CBFA1 activity in human
bone marrow osteoblast progenitors and inhibit osteoblast formation and
differentiation. Blood 106(7): 24722483.
Goldberg M & Smith AJ (2004) Cells and extracellular matrices of dentin and pulp: a
biological basis for repair and tissue engineering. Crit Rev Oral Biol Med 15(1): 13
27.
Grg A, Boguth G, Obermaier C, Posch A & Weiss W (1995) Two-dimensional
polyacrylamide gel electrophoresis with immobilized pH gradients in the first
dimension (IPG-Dalt): the state of the art and the controversy of vertical versus
horizontal systems. Electrophoresis 16(7): 10791086.
Gouwy M, Struyf S, Proost P & Van Damme J (2005) Synergy in cytokine and chemokine
networks amplifies the inflammatory response. Cytokine Growth Factor Rev 16(6):
561580.
Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten P, Robey PG
& Shi S (2002) Stem cell properties of human dental pulp stem cells. J Dent Res 81(8):
531535.
Gronthos S, Mankani M, Brahim J, Robey PG & Shi S (2000) Postnatal human dental pulp
stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U.S.A. 97(25): 13625
13630.
Gu K, Chang S, Ritchie HH, Clarkson BH & Rutherford RB (2000) Molecular cloning of a
human dentin sialophosphoprotein gene. Eur J Oral Sci 108(1): 3542.
Haab BB, Dunham MJ & Brown PO (2001) Protein microarrays for highly parallel
detection and quantitation of specific proteins and antibodies in complex solutions.
Genome Biol 2(2): RESEARCH0004.
Hahn CL, Best AM & Tew JG (2000) Cytokine induction by Streptococcus mutans and
pulpal pathogenesis. Infect Immun 68(12): 67856789.
Hahn CL & Liewehr FR (2007) Relationships between caries bacteria, host responses, and
clinical signs and symptoms of pulpitis. J Endod 33(3): 213219.
Hanks CT, Fang D, Sun Z, Edwards CA & Butler WT (1998) Dentin-specific proteins in
MDPC-23 cell line. Eur J Oral Sci 106 Suppl 1: 260266.
Hao J, He G, Narayanan K, Zou B, Lin L, Muni T, Ramachandran A & George A (2005)
Identification of differentially expressed cDNA transcripts from a rat odontoblast cell
line. Bone 37(4): 578588.
Hao J, Narayanan K, Ramachandran A, He G, Almushayt A, Evans C & George A (2002)
Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue
both in vitro and in vivo. J Biol Chem 277(22): 1997619981.
Hartsough MT & Steeg PS (2000) Nm23/nucleoside diphosphate kinase in human cancers.
J Bioenerg Biomembr 32(3): 301308.
85
Haug SR & Heyeraas KJ (2006) Modulation of dental inflammation by the sympathetic
nervous system. J Dent Res 85(6): 488495.
Henderson B & Wilson M (1996) Cytokine induction by bacteria: beyond
lipopolysaccharide. Cytokine 8(4): 269282.
Heyeraas KJ & Berggreen E (1999) Interstitial fluid pressure in normal and inflamed pulp.
Crit Rev Oral Biol Med 10(3): 328336.
Huang GT, Do M, Wingard M, Park JS & Chugal N (2001) Effect of interleukin-6
deficiency on the formation of periapical lesions after pulp exposure in mice. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod 92(1): 8388.
Hwang YC, Hwang IN, Oh WM, Park JC, Lee DS & Son HH (2008) Influence of TGF-
beta1 on the expression of BSP, DSP, TGF-beta1 receptor I and Smad proteins during
reparative dentinogenesis. J Mol Histol 39(2): 153160.
Jontell M, Okiji T, Dahlgren U & Bergenholtz G (1998) Immune defense mechanisms of
the dental pulp. Crit Rev Oral Biol Med 9(2): 179200.
Kahai S, Vary CP, Gao Y & Seth A (2004) Collagen, type V, alpha1 (COL5A1) is
regulated by TGF-beta in osteoblasts. Matrix Biol 23(7): 445455.
Kaser A, Dunzendorfer S, Offner FA, Ryan T, Schwabegger A, Cruikshank WW,
Wiedermann CJ & Tilg H (1999) A role for IL-16 in the cross-talk between dendritic
cells and T cells. J Immunol 163(6): 32323238.
Kawashima N, Nakano-Kawanishi H, Suzuki N, Takagi M & Suda H (2005) Effect of
NOS inhibitor on cytokine and COX2 expression in rat pulpitis. J Dent Res 84(8):
762767.
Kendziorski C, Irizarry RA, Chen KS, Haag JD & Gould MN (2005) On the utility of
pooling biological samples in microarray experiments. Proc Natl Acad Sci U.S.A.
102(12): 42524257.
Kido MA, Ibuki T, Danjo A, Kondo T, Zhang JQ, Yamaza T, Yamashita Y, Higuchi Y &
Tanaka T (2005) Immunocytochemical localization of the neurokinin 1 receptor in rat
dental pulp. Arch Histol Cytol 68(4): 259265.
Kimura N, Shimada N, Fukuda M, Ishijima Y, Miyazaki H, Ishii A, Takagi Y & Ishikawa
N (2000) Regulation of cellular functions by nucleoside diphosphate kinases in
mammals. J Bioenerg Biomembr 32(3): 309315.
Kirwan RP, Leonard MO, Murphy M, Clark AF & OBrien CJ (2005) Transforming
growth factor-beta-regulated gene transcription and protein expression in human
GFAP-negative lamina cribrosa cells. Glia 52(4): 309324.
Kitagawa M, Ueda H, Iizuka S, Sakamoto K, Oka H, Kudo Y, Ogawa I, Miyauchi M,
Tahara H & Takata T (2007) Immortalization and characterization of human dental
pulp cells with odontoblastic differentiation. Arch Oral Biol 52(8): 727731.
Klatt AR, Nitsche DP, Kobbe B, Macht M, Paulsson M & Wagener R (2001) Molecular
structure, processing, and tissue distribution of matrilin-4. J Biol Chem 276(20):
1726717275.
Kloth JN, Fleuren GJ, Oosting J, de Menezes RX, Eilers PH, Kenter GG & Gorter A (2005)
Substantial changes in gene expression of Wnt, MAPK and TNFalpha pathways
induced by TGF-beta1 in cervical cancer cell lines. Carcinogenesis 26(9): 14931502.
86
Knauper V, Cowell S, Smith B, Lopez-Otin C, OShea M, Morris H, Zardi L & Murphy G
(1997) The role of the C-terminal domain of human collagenase-3 (MMP-13) in the
activation of procollagenase-3, substrate specificity, and tissue inhibitor of
metalloproteinase interaction. J Biol Chem 272(12): 76087616.
Korkmaz Y, Bloch W, Steinritz D, Baumann MA, Addicks K, Schneider K & Raab WH
(2006) Bradykinin mediates phosphorylation of eNOS in odontoblasts. J Dent Res
85(6): 536541.
Kramer IR (1960) The vascular architecture of the human dental pulp. Arch Oral Biol 2:
177189.
Krone JR, Nelson RW, Dogruel D, Williams P & Granzow R (1997) BIA/MS: interfacing
biomolecular interaction analysis with mass spectrometry. Anal Biochem 244(1):
124132.
Lacombe ML, Milon L, Munier A, Mehus JG & Lambeth DO (2000) The human
Nm23/nucleoside diphosphate kinases. J Bioenerg Biomembr 32(3): 247258.
La Fleche RG, Frank RM & Steuer P (1985) The extent of the human odontoblast process
as determined by transmission electron microscopy: the hypothesis of a retractable
suspensor system. J Biol Buccale 13(4): 293305
Langeland K (1987) Tissue response to dental caries. Endod Dent Traumatol 3(4): 149
171.
Lascu I & Gonin P (2000) The catalytic mechanism of nucleoside diphosphate kinases. J
Bioenerg Biomembr 32(3): 237246.
Leeman MF, Curran S & Murray GI (2002) The structure, regulation, and function of
human matrix metalloproteinase-13. Crit Rev Biochem Mol Biol 37(3): 149166.
Levin LG, Rudd A, Bletsa A & Reisner H (1999) Expression of IL-8 by cells of the
odontoblast layer in vitro. Eur J Oral Sci 107(2): 131137.
Lin SK, Kuo MY, Wang JS, Lee JJ, Wang CC, Huang S, Shun CT & Hong CY (2002)
Differential regulation of interleukin-6 and inducible cyclooxygenase gene expression
by cytokines through prostaglandin-dependent and -independent mechanisms in
human dental pulp fibroblasts. J Endod 28(3): 197201.
Linde A (1985) The extracellular matrix of the dental pulp and dentin. J Dent Res 64 Spec
No: 523529.
Linde A & Goldberg M (1993) Dentinogenesis. Crit Rev Oral Biol Med 4(5): 679728.
Liu J, Jin T, Chang S, Ritchie HH, Smith AJ & Clarkson BH (2007) Matrix and TGF-beta-
related gene expression during human dental pulp stem cell (DPSC) mineralization. In
Vitro Cell Dev Biol Anim 43(34): 120128.
Liu J, Jin T, Ritchie HH, Smith AJ & Clarkson BH (2005) In vitro differentiation and
mineralization of human dental pulp cells induced by dentin extract. In Vitro Cell Dev
Biol Anim 41(7): 232238.
Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang
C, Kobayashi M, Horton H & Brown EL (1996) Expression monitoring by
hybridization to high-density oligonucleotide arrays. Nat Biotechnol 14(13): 1675
1680.
87
Lodish H, Berg A, Zipursky SL, Matsudaira P, Baltimore D & Darnel J (2000) Molecular
Cell Biology. 4 New York, US: W. H. Freeman and Company.
Lopez F, Pichereaux C, Burlet-Schiltz O, Pradayrol L, Monsarrat B & Esteve JP (2003)
Improved sensitivity of biomolecular interaction analysis mass spectrometry for the
identification of interacting molecules. Proteomics 3(4): 402412.
Lovschall H, Tummers M, Thesleff I, Fuchtbauer EM & Poulsen K (2005) Activation of
the Notch signaling pathway in response to pulp capping of rat molars. Eur J Oral Sci
113(4): 312317.
Lucchini M, Romeas A, Couble ML, Bleicher F, Magloire H & Farges JC (2002) TGF beta
1 signaling and stimulation of osteoadherin in human odontoblasts in vitro. Connect
Tissue Res 43(23): 345353.
Lukinmaa PL & Waltimo J (1992) Immunohistochemical localization of types I, V, and VI
collagen in human permanent teeth and periodontal ligament. J Dent Res 71(2): 391
397.
Lundgren T & Linde A (1997) Voltage-gated calcium channels and nonvoltage-gated
calcium uptake pathways in the rat incisor odontoblast plasma membrane. Calcif
Tissue Int 60(1): 7985.
MacDougall M, Thiemann F, Ta H, Hsu P, Chen LS & Snead ML (1995) Temperature
sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell
cultures: establishment of clonal odontoblast cell line. Connect Tissue Res 33(13):
97103.
Magloire H, Couble ML, Romeas A & Bleicher F (2004) Odontoblast primary cilia: facts
and hypotheses. Cell Biol Int 28(2): 9399.
Magloire H, Lesage F, Couble ML, Lazdunski M & Bleicher F (2003) Expression and
localization of TREK-1 K+ channels in human odontoblasts. J Dent Res 82(7): 542
545.
Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A & Locati M (2004) The chemokine
system in diverse forms of macrophage activation and polarization. Trends Immunol
25(12): 677686.
88
MAQC Consortium, Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, Baker SC,
Collins PJ, de Longueville F, Kawasaki ES, Lee KY, Luo Y, Sun YA, Willey JC,
Setterquist RA, Fischer GM, Tong W, Dragan YP, Dix DJ, Frueh FW, Goodsaid FM,
Herman D, Jensen RV, Johnson CD, Lobenhofer EK, Puri RK, Schrf U, Thierry-Mieg
J, Wang C, Wilson M, Wolber PK, Zhang L, Amur S, Bao W, Barbacioru CC, Lucas
AB, Bertholet V, Boysen C, Bromley B, Brown D, Brunner A, Canales R, Cao XM,
Cebula TA, Chen JJ, Cheng J, Chu TM, Chudin E, Corson J, Corton JC, Croner LJ,
Davies C, Davison TS, Delenstarr G, Deng X, Dorris D, Eklund AC, Fan XH, Fang H,
Fulmer-Smentek S, Fuscoe JC, Gallagher K, Ge W, Guo L, Guo X, Hager J, Haje PK,
Han J, Han T, Harbottle HC, Harris SC, Hatchwell E, Hauser CA, Hester S, Hong H,
Hurban P, Jackson SA, Ji H, Knight CR, Kuo WP, LeClerc JE, Levy S, Li QZ, Liu C,
Liu Y, Lombardi MJ, Ma Y, Magnuson SR, Maqsodi B, McDaniel T, Mei N,
Myklebost O, Ning B, Novoradovskaya N, Orr MS, Osborn TW, Papallo A, Patterson
TA, Perkins RG, Peters EH, Peterson R, Philips KL, Pine PS, Pusztai L, Qian F, Ren
H, Rosen M, Rosenzweig BA, Samaha RR, Schena M, Schroth GP, Shchegrova S,
Smith DD, Staedtler F, Su Z, Sun H, Szallasi Z, Tezak Z, Thierry-Mieg D, Thompson
KL, Tikhonova I, Turpaz Y, Vallanat B, Van C, Walker SJ, Wang SJ, Wang Y,
Wolfinger R, Wong A, Wu J, Xiao C, Xie Q, Xu J, Yang W, Zhang L, Zhong S, Zong
Y & Slikker W,Jr (2006) The MicroArray Quality Control (MAQC) project shows
inter- and intraplatform reproducibility of gene expression measurements. Nat
Biotechnol 24(9): 11511161.
Mary-Huard T, Daudin JJ, Baccini M, Biggeri A & Bar-Hen A (2007) Biases induced by
pooling samples in microarray experiments. Bioinformatics 23(13): i3138.
McDonnell JM (2001) Surface plasmon resonance: towards an understanding of the
mechanisms of biological molecular recognition. Curr Opin Chem Biol 5(5): 572577.
McLachlan JL, Sloan AJ, Smith AJ, Landini G & Cooper PR (2004) S100 and cytokine
expression in caries. Infect Immun 72(7): 41024108.
McLachlan JL, Smith AJ, Bujalska IJ & Cooper PR (2005) Gene expression profiling of
pulpal tissue reveals the molecular complexity of dental caries. Biochim Biophys Acta
1741(3): 271281.
McLachlan JL, Smith AJ, Sloan AJ & Cooper PR (2003) Gene expression analysis in cells
of the dentine-pulp complex in healthy and carious teeth. Arch Oral Biol 48(4): 273
283.
Miinalainen IJ, Chen ZJ, Torkko JM, Pirila PL, Sormunen RT, Bergmann U, Qin YM &
Hiltunen JK (2003) Characterization of 2-enoyl thioester reductase from mammals.
An ortholog of YBR026p/MRF1'p of the yeast mitochondrial fatty acid synthesis type
II. J Biol Chem 278(22): 2015420161.
Miller JC, Butler EB, Teh BS & Haab BB (2001) The application of protein microarrays to
serum diagnostics: prostate cancer as a test case. Dis Markers 17(4): 225234.
89
Min KS, Kim HI, Chang HS, Kim HR, Pae HO, Chung HT, Hong SH, Shin HI, Hong CU,
Lee SK & Kim EC (2008) Involvement of mitogen-activated protein kinases and
nuclear factor-kappa B activation in nitric oxide-induced interleukin-8 expression in
human pulp cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 105(5): 654
660.
Mitsiadis TA, Fried K & Goridis C (1999) Reactivation of Delta-Notch signaling after
injury: complementary expression patterns of ligand and receptor in dental pulp. Exp
Cell Res 246(2): 312318.
Mitsiadis TA, Hirsinger E, Lendahl U & Goridis C (1998) Delta-notch signaling in
odontogenesis: correlation with cytodifferentiation and evidence for feedback
regulation. Dev Biol 204(2): 420431.
Mitsiadis TA & Rahiotis C (2004) Parallels between tooth development and repair:
conserved molecular mechanisms following carious and dental injury. J Dent Res
83(12): 896902.
Mitsiadis TA, Romeas A, Lendahl U, Sharpe PT & Farges JC (2003) Notch2 protein
distribution in human teeth under normal and pathological conditions. Exp Cell Res
282(2): 101109.
Mjor IA, Sveen OB & Heyeraas KJ (2001) Pulp-dentin biology in restorative dentistry.
Part 1: normal structure and physiology. Quintessence Int 32(6): 427446.
Mollereau C, Simons MJ, Soularue P, Liners F, Vassart G, Meunier JC & Parmentier M
(1996) Structure, tissue distribution, and chromosomal localization of the
prepronociceptin gene. Proc Natl Acad Sci U.S.A. 93(16): 86668670.
Nelson BJ, Danielpour D, Rossio JL, Turpin J & Nacy CA (1994) Interleukin-2 suppresses
activated macrophage intracellular killing activity by inducing macrophages to secrete
TGF-beta. J Leukoc Biol 55(1): 8190.
Netter FH (ed) (2006) Atlas of Human Anatomy. 4 Teterboro, New Jersey, USA: Icon
Learning Systems.
Norlin T, Hilliges M & Brodin L (1999) Immunohistochemical demonstration of
exocytosis-regulating proteins within rat molar dentinal tubules. Arch Oral Biol 44(3):
223231.
OFarrell PZ, Goodman HM & OFarrell PH (1977) High resolution two-dimensional
electrophoresis of basic as well as acidic proteins. Cell 12(4): 11331141.
Ohlmeier S, Kastaniotis AJ, Hiltunen JK & Bergmann U (2004) The yeast mitochondrial
proteome, a study of fermentative and respiratory growth. J Biol Chem 279(6): 3956
3979.
Okamura K, Kobayashi I, Matsuo K, Taniguchi K, Ishibashi Y, Izumi T, Kitamura K &
Sakai H (1995) An immunohistochemical and ultrastructural study of vasomotor
nerves in the microvasculature of human dental pulp. Arch Oral Biol 40(1): 4753.
Okiji T (2002) Pulp As a Connective Tissue. In: Hargreaves KM & Goodis HE (eds)
Seltzer and Benders Dental Pulp. 3 Carol Stream, IL: Quintessence Publishing Co,
Inc, 95122.
90
Oppenheim JJ & Feldmann M (2003) Introduction to the Role of Cytokines in Innate Host
Defense and Adaptive Immunity. In: Oppenheim JJ & Feldmann M (eds) Cytokine
Reference. 3 Academic Press, 320.from
http://apresslp.gvpi.net/apcyto/lpext.dll?f=templates&fn=main-h.htm&2.0
Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, Vega F, Yu N, Wang J, Singh
K, Zonin F, Vaisberg E, Churakova T, Liu M, Gorman D, Wagner J, Zurawski S, Liu
Y, Abrams JS, Moore KW, Rennick D, de Waal-Malefyt R, Hannum C, Bazan JF &
Kastelein RA (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23,
with biological activities similar as well as distinct from IL-12. Immunity 13(5): 715
725.
Palmqvist C, Wardlaw AJ & Bradding P (2007) Chemokines and their receptors as
potential targets for the treatment of asthma. Br J Pharmacol 151(6):725-36.
Palosaari H, Wahlgren J, Larmas M, Ronka H, Sorsa T, Salo T & Tjderhane L (2000) The
expression of MMP-8 in human odontoblasts and dental pulp cells is down-regulated
by TGF-beta1. J Dent Res 79(1): 7784.
Park JE, Chen HH, Winer J, Houck KA & Ferrara N (1994) Placenta growth factor.
Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and
high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem 269(41): 25646
25654.
Piattelli A, Rubini C, Fioroni M, Tripodi D & Strocchi R (2004) Transforming growth
factor-beta 1 (TGF-beta 1) expression in normal healthy pulps and in those with
irreversible pulpitis. Int Endod J 37(2): 114119.
Piecha D, Wiberg C, Morgelin M, Reinhardt DP, Deak F, Maurer P & Paulsson M (2002)
Matrilin-2 interacts with itself and with other extracellular matrix proteins. Biochem J
367(Pt 3): 715721.
Pohto P & Antila R (1968) Demonstration of adrenergic nerve fibres in human dental pulp
by histochemical fluorescence method. Acta Odontol Scand 26(2): 137144.
Qin C, Brunn JC, Cadena E, Ridall A, Tsujigiwa H, Nagatsuka H, Nagai N & Butler WT
(2002) The expression of dentin sialophosphoprotein gene in bone. J Dent Res 81(6):
392394.
Rand TH, Cruikshank WW, Center DM & Weller PF (1991) CD4-mediated stimulation of
human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands
elicit eosinophil migration. J Exp Med 173(6): 15211528.
Ranganathan P, Agrawal A, Bhushan R, Chavalmane AK, Kalathur RK, Takahashi T &
Kondaiah P (2007) Expression profiling of genes regulated by TGF-beta: differential
regulation in normal and tumour cells. BMC Genomics 8: 98.
Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey
A & Chinnaiyan AM (2004) Large-scale meta-analysis of cancer microarray data
identifies common transcriptional profiles of neoplastic transformation and
progression. Proc Natl Acad Sci U.S.A. 101(25): 93099314.
Roberts-Clark DJ & Smith AJ (2000) Angiogenic growth factors in human dentine matrix.
Arch Oral Biol 45(11): 10131016.
91
Sato O, Maeda T, Kobayashi S, Iwanaga T, Fujita T & Takahashi Y (1988) Innervation of
periodontal ligament and dental pulp in the rat incisor: an immunohistochemical
investigation of neurofilament protein and glia-specific S-100 protein. Cell Tissue Res
251(1): 1321.
Sawa Y, Horie Y, Yamaoka Y, Ebata N, Kim T & Yoshida S (2003) Production of colony-
stimulating factor in human dental pulp fibroblasts. J Dent Res 82(2): 96100.
Schena M, Shalon D, Davis RW & Brown PO (1995) Quantitative monitoring of gene
expression patterns with a complementary DNA microarray. Science 270(5235): 467
470.
Shi S & Gronthos S (2003) Perivascular niche of postnatal mesenchymal stem cells in
human bone marrow and dental pulp. J Bone Miner Res 18(4): 696704.
Shi S, Robey PG & Gronthos S (2001) Comparison of human dental pulp and bone
marrow stromal stem cells by cDNA microarray analysis. Bone 29(6): 532539.
Shi Y & Massague J (2003) Mechanisms of TGF-beta signaling from cell membrane to the
nucleus. Cell 113(6): 685700.
Shih JH, Michalowska AM, Dobbin K, Ye Y, Qiu TH & Green JE (2004) Effects of
pooling mRNA in microarray class comparisons. Bioinformatics 20(18): 33183325.
Silverman JD & Kruger L (1987) An interpretation of dental innervation based upon the
pattern of calcitonin gene-related peptide (CGRP)-immunoreactive thin sensory axons.
Somatosens Res 5(2): 157175.
Sloan AJ, Perry H, Matthews JB & Smith AJ (2000) Transforming growth factor-beta
isoform expression in mature human healthy and carious molar teeth. Histochem J
32(4): 247252.
Sloan AJ & Smith AJ (1999) Stimulation of the dentine-pulp complex of rat incisor teeth
by transforming growth factor-beta isoforms 13 in vitro. Arch Oral Biol 44(2): 149
156.
Smith AJ (2003) Vitality of the dentin-pulp complex in health and disease: growth factors
as key mediators. J Dent Educ 67(6): 678689.
Smith AJ (2002) Dentin Formation and Repair. In: Hargreaves KM & Goodis HE (eds)
Seltzer and Benders Dental Pulp. 3 Illinois: Quintessence Publishing Co, Inc, 4162.
Staquet MJ, Durand SH, Colomb E, Romeas A, Vincent C, Bleicher F, Lebecque S &
Farges JC (2008) Different roles of odontoblasts and fibroblasts in immunity. J Dent
Res 87(3): 256261.
Stuart JM, Segal E, Koller D & Kim SK (2003) A gene-coexpression network for global
discovery of conserved genetic modules. Science 302(5643): 249255.
Takahashi K, Kishi Y & Kim S (1982) A scanning electron microscope study of the blood
vessels of dog pulp using corrosion resin casts. J Endod 8(3): 131135.
Takahashi K, Nakanishi T, Yumoto H, Adachi T & Matsuo T (2008) CCL20 production is
induced in human dental pulp upon stimulation by Streptococcus mutans and
proinflammatory cytokines. Oral Microbiol Immunol 23(4): 320327.
Takeda K & Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17(1): 1
14.
92
Tani-Ishii N, Wang CY & Stashenko P (1995) Immunolocalization of bone-resorptive
cytokines in rat pulp and periapical lesions following surgical pulp exposure. Oral
Microbiol Immunol 10(4): 213219.
Ten Cate AR (1994) Structure of the Oral Tissues. In: Ten Cate AR (ed) Oral Histology:
Development, Structure, Function. 5 St. Louis: Mosby, 4557.
Tete S, Mastrangelo F, Scioletti AP, Tranasi M, Raicu F, Paolantonio M, Stuppia L, Vinci
R, Gherlone E, Ciampoli C, Sberna MT & Conti P (2008) Microarray expression
profiling of human dental pulp from single subject. Clin Invest Med 31(2): E5561.
Tjderhane L, Salo T, Larjava H, Larmas M & Overall CM (1998) A novel organ culture
method to study the function of human odontoblasts in vitro: gelatinase expression by
odontoblasts is differentially regulated by TGF-beta1. J Dent Res 77(7): 14861496.
Tokuda M, Sakuta T, Fushuku A, Torii M & Nagaoka S (2001) Regulation of interleukin-6
expression in human dental pulp cell cultures stimulated with Prevotella intermedia
lipopolysaccharide. J Endod 27(4): 273277.
Tran-Hung L, Mathieu S & About I (2006) Role of human pulp fibroblasts in angiogenesis.
J Dent Res 85(9): 819823.
Trowbridge HO (2002) Histology of Pulpan Inflammation. In: Hargreaves KM & Goodis
HE (eds) Seltzer and Benders Dental Pulp. 3 Illinois: Quintessence Publishing Co,
Inc, 227245.
Tsuzaki M, Yamauchi M & Mechanic GL (1990) Bovine dental pulp collagens:
characterization of types III and V collagen. Arch Oral Biol 35(3): 195200.
Tziafas D (2004) The future role of a molecular approach to pulp-dentinal regeneration.
Caries Res 38(3): 314320.
Tziafas D, Smith AJ & Lesot H (2000) Designing new treatment strategies in vital pulp
therapy. J Dent 28(2): 7792.
Uddman R, Kato J, Cantera L & Edvinsson L (1998) Localization of neuropeptide Y Y1
receptor mRNA in human tooth pulp. Arch Oral Biol 43(5): 389394.
Valsecchi C, Ghezzi C, Ballabio A & Rugarli EI (1997) JAGGED2: a putative Notch
ligand expressed in the apical ectodermal ridge and in sites of epithelial-mesenchymal
interactions. Mech Dev 69(12): 203207.
Veerayutthwilai O, Byers MR, Pham TT, Darveau RP & Dale BA (2007) Differential
regulation of immune responses by odontoblasts. Oral Microbiol Immunol 22(1): 5
13.
Velculescu VE, Zhang L, Vogelstein B & Kinzler KW (1995) Serial analysis of gene
expression. Science 270(5235): 484487.
Verstak B, Hertzog P & Mansell A (2007) Toll-like receptor signalling and the clinical
benefits that lie within. Inflamm Res 56(1): 110.
Vilcek J (2002) Cytokines Engaged in Antiviral Action, Macrophage Activation,
Angiogenesis, and Regulation of Cell Growth and Differentiation. In: Oppenheim JJ
& Feldmann M (eds) Cytokine Reference. 3 Academic Press, 615625.from
http://apresslp.gvpi.net/apcyto/lpext.dll?f=templates&fn=main-h.htm&2.0
Vongsavan N, Matthews RW & Matthews B (2000) The permeability of human dentine in
vitro and in vivo. Arch Oral Biol 45(11): 931935.
93
Wakisaka S, Nishikawa S, Ichikawa H, Matsuo S, Takano Y & Akai M (1985) The
distribution and origin of substance P-like immunoreactivity in the rat molar pulp and
periodontal tissues. Arch Oral Biol 30(1112): 813818.
Wang X, Jia S, Meyer L, Xiang B, Chen LY, Jiang N, Moreno C, Jacob HJ, Ghosh S &
Hessner MJ (2006) Comprehensive quality control utilizing the prehybridization third-
dye image leads to accurate gene expression measurements by cDNA microarrays.
BMC Bioinformatics 7: 378.
Wang X, Jiang N, Feng X, Xie Y, Tonellato PJ, Ghosh S & Hessner MJ (2003) A novel
approach for high-quality microarray processing using third-dye array visualization
technology. IEEE Trans Nanobioscience 2(4): 193201.
Wiberg C, Klatt AR, Wagener R, Paulsson M, Bateman JF, Heinegard D & Morgelin M
(2003) Complexes of matrilin-1 and biglycan or decorin connect collagen VI
microfibrils to both collagen II and aggrecan. J Biol Chem 278(39): 3769837704.
Xiao S, Yu C, Chou X, Yuan W, Wang Y, Bu L, Fu G, Qian M, Yang J, Shi Y, Hu L, Han
B, Wang Z, Huang W, Liu J, Chen Z, Zhao G & Kong X (2001) Dentinogenesis
imperfecta 1 with or without progressive hearing loss is associated with distinct
mutations in DSPP. Nat Genet 27(2): 201204.
Xie L, Law BK, Aakre ME, Edgerton M, Shyr Y, Bhowmick NA & Moses HL (2003)
Transforming growth factor beta-regulated gene expression in a mouse mammary
gland epithelial cell line. Breast Cancer Res 5(6): R18798.
Yamada Y, Fujimoto A, Ito A, Yoshimi R & Ueda M (2006) Cluster analysis and gene
expression profiles: a cDNA microarray system-based comparison between human
dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for
tissue engineering cell therapy. Biomaterials 27(20): 37663781.
Yang LC, Huang FM, Lin CS, Liu CM, Lai CC & Chang YC (2003a) Induction of
interleukin-8 gene expression by black-pigmented Bacteroides in human pulp
fibroblasts and osteoblasts. Int Endod J 36(11): 774779.
Yang LC, Tsai CH, Huang FM, Liu CM, Lai CC & Chang YC (2003b) Induction of
interleukin-6 gene expression by pro-inflammatory cytokines and black-pigmented
Bacteroides in human pulp cell cultures. Int Endod J 36(5): 352357.
Zehnder M, Delaleu N, Du Y & Bickel M (2003) Cytokine gene expression--part of host
defence in pulpitis. Cytokine 22(34): 8488.
Zhang C, Chang J, Sonoyama W, Shi S & Wang CY (2008) Inhibition of human dental
pulp stem cell differentiation by Notch signaling. J Dent Res 87(3): 250255.
Zhu H, Bilgin M & Snyder M (2003) Proteomics. Annu Rev Biochem 72: 783812.
94
List of original publications
The thesis is based on following articles, which are referred to in the text by
Roman numerals:
I Pkknen V, Ohlmeier S, Bergmann U, Larmas M, Salo T & Tjderhane L (2005)
Analysis of gene and protein expression in healthy and carious tooth pulp with cDNA
microarray and two-dimensional gel electrophoresis. Eur J Oral Sci 113: 369379.
II Pkknen V, Vuoristo JT, Salo T & Tjderhane L (2008) Comparative gene
expression profile analysis between native human odontoblasts and pulp tissue. Int
Endod J 41:117127.
III Pkknen V, Vuoristo J, Salo T & Tjderhane L (2007) Effects of TGF-beta1 on
interleukin profile of human dental pulp and odontoblasts. Cytokine 40: 4451.
IV Pkknen V, Bleicher F, Carrouel F, Vuoristo JT, Salo T, Wappler I, Couble ML,
Magloire H, Peters H & Tjderhane L (2009) General expression profiles of human
native odontoblasts and pulp-derived cultured odontoblast-like cells are similar but
reveal differential neuropeptide expression levels. Arch Oral Biol 54: 5562.
Reprinted with permission from Wiley-Blackwell (I, II) and Elsevier (III, IV).
Original publications are not included in the electronic version of the dissertation.
95
96
ACTA UNIVERSITATIS OULUENSIS
SERIES D MEDICA
987. Majalahti, Theresa (2008) Mechanisms of cardiac chamber-specific gene

expression of natriuretic peptides
988. Kaakinen, Hanna (2008) Approaches to improving brain protection in cardiac and
aortic surgery. An experimental study in a porcine model with hypertonic saline
dextran, levosimendan, leukocyte depleting filter and different acid base
management strategies
989. Korhonen, Ilkka (2008) Thermal, hormonal and cardiovascular responses to
single and repeated nonhypothermic cold exposures in man
990. Saarenp, Ismo (2008) Extracapsular hip fracturesaspects of intramedullary
and extramedullary fixation
991. Rantavuori, Kari (2008) Aspects and determinants of childrens dental fear
992. Kunnari, Anne (2008) Genetic, epidemiological and cell culture studies on human
resistin
993. Tahkola, Jenni (2008) Collagen XIII in cardiovascular development and
tumorigenesis
994. Tuomisto, Anne (2008) The role of collagen XIII in B-cell lymphoma
development, and characterization of its biosynthesis and tissue distribution
995. Serlo, Kaijaleena (2008) University students attitudes towards HIV/AIDS in
Finland and in Kenya
996. Erkko, Hannele (2008) TOPBP1, CLSPN and PALB2 genes in familial breast cancer
susceptibility
997. Peltonen, Tuomas (2008) Endothelial factors in the pathogenesis of aortic valve
stenosis
998. Leinonen, Jukka (2008) Carbonic anhydrase isoenzyme VI: distribution, catalytic
properties and biological significance
999. Ulvila, Johanna (2008) Functional genomic analysis of the Drosophila immune
response. Identification of genes essential for phagocytosis, viral defense and NF-
B signaling
1000. Lajunen, Taina (2008) Persistent Chlamydia pneumoniae infection, inflammation
and innate immunity
1001. Leinonen, Pekka (2008) Calcium signaling in epithelium. Special focus on Hailey-
Hailey and Darier diseases, neurofibromatosis 1 and transition cell carcinoma
Book orders: Distributed by

OULU UNIVERSITY PRESS OULU UNIVERSITY LIBRARY
P.O. Box 8200, FI-90014 P.O. Box 7500, FI-90014
University of Oulu, Finland University of Oulu, Finland
OULU 2009
D 1002
D 1002
U N I V E R S I T Y O F O U L U P. O. B . 7 5 0 0 F I - 9 0 0 1 4 U N I V E R S I T Y O F O U L U F I N L A N D
ACTA U N I V E R S I T AT I S O U L U E N S I S
ACTA
A C TA U N I V E R S I TAT I S O U L U E N S I S
S E R I E S E D I T O R S
Virve Pkknen
D MEDICA
ASCIENTIAE RERUM NATURALIUM

EXPRESSION PROFILING
Virve Pkknen
Professor Mikko Siponen
BHUMANIORA
University Lecturer Elise Krkkinen
OF HUMAN PULP TISSUE
CTECHNICA
AND ODONTOBLASTS
Professor Hannu Heusala IN VIVO AND IN VITRO
DMEDICA
ESCIENTIAE RERUM SOCIALIUM

Senior Researcher Eila Estola
FSCRIPTA ACADEMICA
Information officer Tiina Pistokoski
GOECONOMICA
University Lecturer Seppo Eriksson
EDITOR IN CHIEF
PUBLICATIONS EDITOR
Publications Editor Kirsti Nurkkala FACULTY OF MEDICINE,
INSTITUTE OF DENTISTRY,
UNIVERSITY OF OULU;
ISBN 978-951-42-9004-6 (Paperback) INSTITUTE OF DENTISTRY,
ISBN 978-951-42-9005-3 (PDF) UNIVERSITY OF HELSINKI
ISSN 0355-3221 (Print)

Isbn 9789514290053

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Isbn 9789514290053

Uploaded by

Copyright:

Available Formats

OULU 2009

ASCIENTIAE RERUM NATURALIUM

ESCIENTIAE RERUM SOCIALIUM

EXPRESSION PROFILING OF HUMAN

Academic dissertation to be presented, with the assent of

ISBN 978-951-42-9004-6 (Paperback)

OULU UNIVERSITY PRESS

Keywords: dental pulp, gene expression profiling, humans, odontoblasts, TGF-

Oulu, December 2008 Virve Pkknen

2.1 Structure of the tooth

Fig. 1. Tooth structure modified from Netter 2006.

2.2 Dentin-pulp complex

Dentin is a mineralized connective tissue, and its characteristics are largely

2.2.4 Inflammatory response of the dentin-pulp complex

Invasion of bacteria or bacteria-derived factors into the pulp is a major etiologic

2.3 Cytokines in the dentin-pulp complex

2.3.1 Inflammatory cytokines in the dentin-pulp complex

Multiple inflammatory cytokines have been detected in the dentin-pulp complex

Cytokine Function Target

Inflammatory cytokines in healthy pulp

Inflammatory cytokines in healthy odontoblasts

Cytokine response of pulp to caries

The expression of various inflammatory cytokines, e.g. several ILs and

Table 2. Cytokines upregulated in inflamed pulp tissue in vivo or in response to

Cytokine In vivo In vitro Reference

Bacterial antigens induce the production of proinflammatory cytokines by

Table 3. Cytokines upregulated in inflamed odontoblasts in vivo or in response to

Cytokine In vivo In vitro Reference

Neuropeptides are peptides secreted by neurons and function as signaling

2.5 Transcriptome and proteome of the dentin-pulp complex

High-throughput methods to study transcriptome include e.g. DNA microarray,

A DNA microarray consists of oligonucleotides or cDNA fragments that are either

In any microarray experiment image analysis, signal adjustment and data

Suppression subtractive hybridization (SSH)

Serial analysis of gene expression (SAGE)

Massive parallel signature sequencing (MPSS)

MPSS (Brenner et al. 2000) is a powerful sequencing method generating millions

In 2-D electrophoresis, proteins are first separated in one direction by isoelectric

In antibody-based arrays antibodies are spotted onto a surface, such as a glass

Biomolecular interaction analysis/mass spectrometry (BIA/MS)

BIA/MS is a method in which binding of an analyte in solution to a surface-

2.5.3 Transcriptomics and proteomics in pulp biology

4.1.1 Collection of mature native teeth and native odontoblast and

4.1.3 Cultured odontoblast-like cells (IV)

The odontoblast-like cells were cultured as previously described (Couble et al.

4.2 Extraction of RNA and proteins (IIV)

4.3 Large-scale analysis of gene expression

4.3.1 cDNA microarray (I)

4.3.2 GeneChip microarray (II, III, IV)

GeneChip microarrays (Affymetrix, Santa Clara, CA, USA) were performed

Data analysis (II, III, IV)

Data downloaded from Gene Expression Omnibus (II)

4.4 RT-PCR (I, II, III)

4.5 Real-time quantitative PCR (IV)

4.6.1 2-D electrophoresis and mass spectrometry (MS) (I)

4.6.2 Antibody array (III)

4.7 Western blot (II)

4.8 Immunofluorescence (IV)

Cryostat sections (10 m) were prepared as described previously (Allard et al.

5.1.1 The effects of caries on the mRNA profile of pulp tissue

After separation by 2-D electrophoresis, approximately 400 protein spots were

To determine the repeatability of the Affymetrix microarrays, expression profile

5.3 The effects of TGF-1 on the general expression profile of pulp