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Original Article
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Bull Tokyo Dent Coll (2006) 47(3): 117–124
Morphohistological Change and Expression of HSP70,
Osteopontin and Osteocalcin mRNAs in Rat Dental Pulp
Cells with Orthodontic Tooth Movement
Satoshi Shigehara, Kenichi Matsuzaka* and Takashi Inoue*
Department of Clinical Pathophysiology, Tokyo Dental College,
1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan
* Oral Health Science Center HRC7,
Department of Clinical Pathophysiology, Tokyo Dental College,
1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan
Received 8 September, 2006/Accepted for publication 4 October, 2006
Abstract
Morphological change and expression of osteopontin, osteocalcin, and HSP70
mRNAs in rat dental pulp cells with experimental orthodontic tooth movement were
investigated. Elastic rubber blocks, 0.65mm in thickness, were inserted between the
maxillary first and second molars in rats. In addition to morphological observations of
HE staining and TUNEL staining at days 3, 7, 14 and 28 after insertion of elastic rubber
blocks, expression of HSP70, osteopontin and osteocalcin mRNAs was also analyzed using
quantitative RT-PCR with a LightCycler™. Morphologically, proliferation and vasodilation
of capillaries was evident in the pulp at days 3 and 7, and a sparse odontoblast layer and
apoptosis in the pulp were observed at days 7 and 14 after rubber block insertion.
Expression of HSP70, osteopontin and osteocalcin mRNAs in the experimental groups
was higher than that in the control group at all time points. This suggests that orthodontic
tooth movement causes degenerative changes and apoptosis in pulp cells, while pulp
homeostasis is maintained at the genetic level.
Key words: Tooth movement—HSP70—Osteopontin—Osteocaclin—mRNA
significant (27.4%) depression in pulpal res-
piratory rate when a tooth undergoes orth-
odontic movement
7)
. Kucukkeles reported
that vascular degeneration was the main
change in pulp during orthodontic tooth
movement
11)
. Although vascular blood flow
affects pulp tissue during degenerative change,
homeostasis of the pulp also occurs during
Introduction
Orthodontic tooth movement involves
dynamic changes in the periodontal ligament
and alveolar bone. Tissue change during
orthodontic loading is not only evoked
in periodontal ligament, but also in pulp
tissue
7,8,11,17,18,29,33)
. Hamersky demonstrated a
This paper is a thesis submitted to the Graduate School of Tokyo Dental College.
118
orthodontic tooth movement. Orthodontic
tooth movement also affects dental pulp in a
way similar to surgical or chemical stimulation.
It has been demonstrated that dental pulp
tissue has the ability to form dentin or
osteodentin during wound healing induced
either by surgical or chemical stimulation
2,25,27)
.
The formation of dentin or osteodentin in the
dental pulp is known to be one of the reac-
tions of homeostasis. Bone-related protein is
related to the calcification of pulp
21)
, and hard
tissue formation is regulated by the activity of
pulp cells
1,28)
. Heat shock protein 70 (HSP70),
which serves as a molecular chaperone to
maintain homeostasis, is expressed physio-
logically in response to stimulation
24)
. Chen et
al. reported that HSP70 expression in pulp
increased during reparative dentinogenesis
4)
.
The induction of this stress protein has also
been investigated using rat nerve cells or
gerbil brain under hypoxic conditions
19,31,32)
.
Furthermore, apoptosis plays an important
role in homeostasis
6,12)
. However, little is known
about the homeostatic mechanism of dental
pulp cells during orthodontic tooth movement.
Therefore, the aim of this study was to
evaluate morphological changes such as pro-
grammed cell death by using TdT-mediated
dUTP-biotin nick end labeling (TUNEL)
method and expression of osteopontin, osteo-
calcin and HSP70 mRNAs in rat dental pulp
cells during experimental orthodontic tooth
movement.
Materials and Methods
1. Animals
Thirty-four male Sprague-Dawley rats weigh-
ing 200–250g each (7–8 weeks old), were used
in this study. The animals given water and
food kneaded with water, and were housed
in a room with a 12-hr light/dark cycle; they
were acclimatized for 1 week before com-
mencement of the experiments. The study
protocol was designed in accordance with
the “Principles of Laboratory Animal Care”
(NIH publication No. 86-23, revised 1985) and
relevant national laws.
2. Experimental design
After administration of general anesthesia
using sodium thiopental, elastic rubber blocks,
0.65ן0.65ן1.5mm, were inserted between
the maxillary first and second molars bilater-
ally in each rat, according to the method of
Waldo and Rothblatt (Fig. 1)
34)
. As a control
group, we also used rats with no rubber block
insertion.
For histological observations, 10 of the rats
were divided into 4 experimental groups and
a control group consisting of 2 animals each.
On days 3, 7, 14 and 28 after insertion of
the rubber block (and in the control group),
the animals were anesthetized with sodium
thiopental and perfused intracardially with
4% paraformaldehyde in 0.2M phosphate
buffer (pH7.3). Each animal was checked to
ensure that the rubber blocks remained in
place during the examination period. Maxillae
were removed and stored in the same fixative
at 4°C for 2 days. Each specimen was deminer-
alized in 10% EDTA for 2 weeks, and then
rinsed in 0.2M phosphate buffered saline
(PBS), dehydrated with graded ethanols, and
embedded in paraffin. Sections in the mesio-
distal sagittal plane at a thickness of approxi-
mately 5␮m were collected on glass slides,
stained with hematoxylin and eosin, and
then observed by microscopy. For TUNEL,
the sections were incubated with 16.2␮g/ml
proteinase K solution (in 10mM Tris-HCl
buffer, pH7.4) for 20min following depar-
Shigehara S et al.
Fig. 1 Schematic drawing of placement of rubber blocks
Rubber blocks were inserted between 1st and
2nd molars of maxillae. M: maxilla, P: pulp
119
affinization with xylene. After washing for
5min, the sections were incubated in TdT
solution (TdT buffer, TdT, Biotin-16-dUTP)
for 90min at 37°C, and the reaction was
terminated by incubating with termination
buffer for 30min. After the sections were
washed with water and PBS, they were stained
with 3,3Ј-diaminobenzidine for 5min, washed
with distilled water, counterstained in hema-
toxylin, and coverslipped. Finally, the pulp of
the distal root was observed histologically.
We then evaluated osteocalcin, osteopontin
and HSP70 mRNAs in 3 further experimental
groups and a control group consisting of
6 animals each. On days 3, 7 and 14 after
insertion of the rubber block, the animals
were anesthetized with an overdose of sodium
thiopental and checked to ensure that the
rubber blocks remained in place during the
examination period. The first molars were
extracted, and the periodontal ligament was
removed mechanically (using a knife observed
through a loupe). The periodontal ligament
was then immersed in a solution of sodium
hypochlorite. Following washing with PBS,
the teeth were broken using a dental chisel
and a mallet. Each pulp was then carefully
collected into an Eppendorf tube with a
dental explorer and a dental scaler using a
microscope. Six animals without rubber block
insertions were used as controls. For quan-
titative analysis of osteopontin, osteocalcin
and HSP70 mRNA expression, total RNA
was extracted using Isogen reagent (Nippon
Gene, Japan) according to the manufacturer’s
instructions. Briefly, cells were homogenized
and solubilized in the Isogen/chloroform
solution at 4°C. Supernatants were obtained
following centrifugation at 12,000ןg for
20min at 4°C. The precipitates were obtained
by decantation and washed with 75% ethanol.
The RNA pellets were then dissolved in
RNase-free water, and preserved at מ20°C
until used. Using the extracted RNA as a
template, reverse transcription reactions were
conducted with an RT-PCR kit (RNA-PCR
kit ver. 2.1, Takara Biomedicals, Japan) to
synthesize cDNA. Quantitative PCR was then
conducted using primers for osteopontin,
osteocalcin, and HSP70 with a LightCycler
®
using double-stranded DNA dye SYBR Green
I (Roche Diagnostics, Germany). The PCR
conditions and primer sequences used in the
LightCycler™ are shown in Table 1. Quan-
tification was performed by comparing the
levels obtained with standard samples. In the
present study, the concentrated solutions of
cDNA in the unstimulated samples were 0.2,
0.5, 1.0 and 2.0␮l. PCR with the LightCycler™
was carried out over 40 cycles. After PCR
amplification, melting curve analyses were
also performed to confirm the absence of
the primer dimer in the PCR products. The
ratios of osteopontin mRNA expression were
adjusted according to the value of the house-
keeping gene, GAPDH. Four independent
experiments were performed for all assays of
cell behavior; two specimens of each sample
Dental Pulp Changes during Tooth Movement
Table 1 PCR primers used for LightCycler™-assisted PCR analysis
Target cDNA Primer sequence (5Ј to 3Ј) PCR condition Product size
Osteopontin Forward; CTC GGA GGA GAA GGC GCA TTA 40 cycles
207bp
Reverse; CCA TCG TCA TCG TCG TCG TCA (95°C 10sec, 60°C 5sec, 72°C 12sec)
Osteocalcin Forward; GGT GCA AAG CCC AGC GAC TCT 40 cycles
199bp
Reverse; GGA AGC CAA TGT GGT CCG CTA (95°C 10sec, 60°C 5sec, 72°C 12sec)
HSP70 Forward: GTG TGC AAC CCG ATC ATC AG 40 cycles
180bp
Reverse; CAC CAG CAG CCA TCA AGA GT (95°C 10sec, 60°C 10sec, 72°C 7sec)
GAPDH Forward: TGC CCA GAA CAT CAT CCC TG 40 cycles
307bp
Reverse; TCA GAT CCA CGA CGG ACA CA (95°C 10sec, 60°C 10sec, 72°C 12sec)
120
type were used in each experiment.
3. Statistical analysis
The results obtained at each experimental
time point were compared with those of the
control group using the Mann-Whitney U-test.
Results
1. Histological observations (Fig. 2a–f)
In the control group, a layer of densely
packed odontoblasts was observed close to
the dentin, under which there was a cell-rich
layer. A few undilated vessels were observed in
the center of the pulp (Fig. 2a). At days 3 and
7 after rubber block insertion, multiplication
and vasodilation of capillary vessels were
distinguished (Fig. 2b, c). Furthermore, a
sparse odontoblast layer was recognized at
days 7 and 14 after insertion, and cells in both
the odontoblast layer and underneath it were
degenerated and vacuolated (Fig. 2c, d). On
the other hand, at day 28 after insertion,
morphologically, no vasodilation was observed,
and the odontoblasts lay in a densely-packed
arrangement (data not shown).
No cells positive for TUNEL in the pulp were
found in the controls (data not shown). At
days 7 and 14 after insertion, TUNEL-positive
cells, showing separation, condensation and
fragmentation of chromatin, were found in
the pulp underlying the odontoblast layer
and central root area (Fig. 2e, f, g, h).
2. Expression of osteopontin, osteocalcin
and HSP70 mRNA
Expression of osteopontin mRNA in the
experimental groups was approximately 1.3
times higher than that in the control group at
days 3, 7 and 14 after insertion of the rubber
block (Fig. 3). Osteopontin mRNA expression
at day 3 after insertion was significantly higher
than that in the controls (pϽ0.01). Osteo-
calcin mRNA expression at day 3 after inser-
tion was approximately 2 times higher than
that in the controls (pϽ0.01). At day 14 after
insertion, expression of osteocalcin mRNA
showed a decrease to a level similar to that
of the controls (Fig. 4). Furthermore, HSP70
mRNA expression at day 3 after insertion was
2.8 times higher than that of the controls, and
at days 7 and 14 was approximately 2 times
higher (pϽ0.01) (Fig. 5).
Discussion
A number of phenomena such as vacuolar
degeneration of odontoblasts, bulging of
microvessels, and production of denticles have
all been reported as effects of orthodontic
tooth movement on pulp
26)
. In this experiment,
we found vacuolar degeneration, a sparse
odontoblast layer, and dilatation of capillary
vessels, as reported in earlier studies
3,25)
.
During tooth movement, pulpal changes may
occur primarily due to alterations in blood
vessels in the periodontium and those entering
the pulp
25)
. There have been several reports
on circulatory disturbance in dental pulp
during orthodontic tooth movement
5,23,30,35)
,
so it is well known that orthodontic tooth
movement affects dental pulp cells when
blood circulation is disturbed. Apoptosis is an
essential mechanism during development and
normal tissue injury
22)
. Since the concept of
apoptosis was first postulated, many studies on
apoptosis in the oral region have investigated
periodontal tissues during tooth eruption
9,10,13)
.
The TUNEL method, which is based on the
specific binding of TdT to 3Ј-OH of DNA, has
been reported as an in situ labeling method.
Rana et al. demonstrated that TUNEL-positive
cells were found in pulp
20)
. In this study,
TUNEL-positive cells were also observed out-
side the coronal area in the pulp. This study
demonstrated that orthodontic tooth move-
ment evoked not only degenerative change,
but also apoptosis in pulp cells. Amemiya
studied pulp cell responses during hypoxia in
vitro, and showed that alkaline phosphatase
activity in pulp cells under hypoxic conditions
was significantly higher than in the controls
1)
.
Further, Wong et al. reported that orthodontic
tooth movement caused vasodilation in the
pulp
35)
. Moreover, it is known that local blood
circulatory disturbances affect calcification in
Shigehara S et al.
121 Dental Pulp Changes during Tooth Movement
Fig. 2 Histological observations of dental pulp
bar: a–e, g: 500␮m, f, h: 100␮m, a: H-E staining of control group, b: H-E staining at day 3 after rubber
block insertion, c: H-E staining at day 7 after rubber block insertion, d: H-E staining at day 14 after rubber
block insertion, e: TUNEL staining at day 7 after rubber block insertion (arrow: TUNEL-positive cell),
f: high magnification of box area of figure e, g: TUNEL staining at day 14 after rubber block insertion
(arrow: TUNEL-positive cell), h: high magnification of box area of figure g.
122
14, osteocalcin mRNA expression showed a
decrease, while osteopontin mRNA expression
remained high. Osteocalcin is involved in
matrix mineralization, while osteopontin is
involved in differentiation of odontoblasts.
Morphologically, in this study, we found
vacuolar degeneration of odontoblasts, which
suggests that pulp cells differentiate into
odontoblasts to compensate for this regressive
change. HSP70 is a molecular chaperone
needed to maintain homeostasis
24)
. Here,
HSP70 mRNA expression during orthodontic
tooth movement was higher than that in
the controls. This result differs from that of
an in vitro experiment where HSP70 mRNA
expression showed no change with hypoxia
1)
.
Amemiya demonstrated that HSP70 expres-
sion increased after reoxygenation
1)
. In their
in vitro experiment, only fibroblast-like pulp
cells were selected, but the reactions of the
dental pulp in our in vivo experiment involved
a complex of interacting factors. Chen et al.
suggested that HSP70 plays an important role
as a molecular chaperone during reparative
dentin formation
4)
. The increase of HSP70
mRNA expression found in this study supports
Chen’s hypothesis
4)
.
In conclusion, these results suggest that
expression of HSP70, osteopontin, and osteo-
calcin mRNA functions as a homeostatic
mechanism to compensate for pulpal cell
changes occurring during orthodontic force
as a result of circulatory disturbance.
Fig. 5 Quantitative reverse transcription-polymerase
chain reaction analyses of HSP70 mRNA in
dental pulp
Data are shown as folds of corresponding con-
trol group. *: significant difference (pϽ0.01)
Fig. 3 Quantitative reverse transcription-polymerase
chain reaction analyses of osteopontin mRNA
in dental pulp
Data are shown as folds of corresponding con-
trol group. *: significant difference (pϽ0.01)
Fig. 4 Quantitative reverse transcription-polymerase
chain reaction analyses of osteocalcin mRNA
in dental pulp
Data are shown as folds of corresponding con-
trol group. *: significant difference (pϽ0.01)
Shigehara S et al.
tissue. In this study, osteopontin and osteo-
calcin mRNA expression was higher in the
experimental groups than in the control
group. Osteopontin and osteocalcin are well-
known markers of bone-related cell differ-
entiation
14–16,21)
. In particular, osteopontin is a
marker of osteoblast or odontoblast differ-
entiation
21)
. Rashid et al. suggests that, with
orthodontic tooth movement, disturbance of
blood flow in the dental pulp incurs hypoxic
conditions, which causes pulpal calcification,
formation of denticles and differentiation
of pulp cells into odontoblasts
21)
. In this
study, although no pulpal calcification was
observed, mRNA expression-related calcifica-
tion increased. On the other hand, at day
123
Acknowledgements
We would like to thank Dr. Eitoyo Kokubu
and other members of the Department of
Clinical Pathophysiology, Tokyo Dental Col-
lege for their technical assistance.
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Reprint requests to:
Dr. Kenichi Matsuzaka
Department of Clinical Pathophysiology,
Tokyo Dental College,
1-2-2 Masago, Mihama-ku,
Chiba 261-8502, Japan
Tel: +81-43-270-3581
Fax: +81-43-270-3583
E-mail: matsuzak@tdc.ac.jp
Shigehara S et al.