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Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College,
Available from http://ir.tdc.ac.jp/
Title
Effect of basic fibroblast growth factor on root
resorption after delayed autotransplantation of
tooth in dogs
Author(s)
Alternative
Shiratani, S; Ota, M; Fujita, T; Seshima, F;
Yamada, S; Saito, A
JournalOral surgery, oral medicine, oral pathology and
oral radiology, 114(2): e14-e21
URL http://hdl.handle.net/10130/2932
Right
1
Ms. Ref. No.: TRIPLEO-D-11-01246
Revised
Effect of basic fibroblast growth factor on root resorption following
delayed autotransplantation of tooth in dog
Satoru Shiratani, DDS, a Mikio Ota, DDS, PhD b Takahisa Fujita, DDS, PhD, c
Fumi Seshima, DDS, PhD, c Satoru Yamada, DDS, PhD,d Atsushi Saito, DDS,
PhD e
aPhD candidate, Department of Periodontology, Tokyo Dental College, Chiba, Japan bSenior Assistant Professor, Department of Periodontology, Tokyo Dental College,
Chiba, Japan cAssistant Professor, Department of Periodontology, Tokyo Dental College, Chiba,
Japan dProfessor Emeritus, Department of Periodontology, Tokyo Dental College, Chiba,
Japan eProfessor and Chair, Department of Periodontology, Tokyo Dental College, Chiba,
Japan
Corresponding author; Atsushi Saito, DDS, PhD
Department of Periodontology, Tokyo Dental College,
1-2-2 Masago, Mihama-ku, Chiba,
261-8502 Japan
Phone: +81-43-270-3952, Fax: +81-43-270-3955, E-mail: atsaito@tdc.ac.jp
The authors report no conflicts of interest.
2
Word count:
Abstract: 150
Complete manuscript: 3319
Number of references: 47
Number of figures: 3
Number of tables: 2
3
Abstract
Objectives. To investigate the effect of basic fibroblast growth factor (FGF-2) on root
resorption following delayed autotransplantation in dog.
Study design. Mandibular second and third premolars of beagle dogs were extracted to
create sites for autotransplantation. After two months, in the experimental sites, the first
and fourth mandibular premolars were extracted and air-dried prior to
autotransplantation with the application of recombinant FGF-2, while control sites
received teeth without FGF-2. At 2, 4, or 8 weeks after surgery, the animals were
sacrificed and specimens collected and processed for histological examination.
Results. Autotransplantation with FGF-2 yielded formation of new periodontal
ligament-like tissues with inserting collagen fibers, associated cementum and bone. The
occurrence of replacement resorption in the FGF-2 treated group was significantly
lower than that in the control group (P < .01).
Conclusion. It was demonstrated that topical application of FGF-2 reduced the
occurrence of ankylosis and root resorption following delayed autotransplantation in
this experimental model.
4
Key words: Basic fibroblast growth factor; Autotransplantation; Histopathology; Wound
healing; in vivo
5
Introduction
Autogenous tooth transplantation can be an alternative treatment modality, especially in
situations where dental implants and other prosthetic replacements are
contraindicated.1-4 When treated with adequate indications, autotransplantation offers
a fast and economically viable option for replacing missing teeth 2,5-7
Root resorption, the most common complication accompanying autotransplantation,
leads to poor esthetics, the tilting of adjacent teeth, loss of function, and permanent
tooth loss.8-10 Occurrence of replacement resorption is influenced by factors such as
the extra-alveolar period11, preservative solution12, pulpal revascularization13, and
microbial contamination.14 Treatment, often complex, time-consuming, and expensive,
requires a multidisciplinary approach including endodontic, periodontal, surgical, and
orthodontic treatment, as well as esthetic coronal restoration.15
Root resorption may be transitory or progressive. Replacement resorption is
followed by ankylosis, and the root surface is gradually replaced with bone.16 The
probability of successful autotransplantation increases when an avulsed tooth is
6
immediately transplanted or stored in a solution that preserves periodontal ligament
(PDL) on the root.11, 14, 17, 18 PDL cells are easily injured under stressful conditions
such as variable pH, osmotic pressure or dehydration, and favorable healing of the PDL
depends on the quantity of viable cells preserved on the root.1 Andreasen11 showed that
only 21% of normal periodontium was hisopathologically observed when extracted
tooth was left in a dry condition for 60 min. prior to tooth replantation in monkeys.
Autotransplantation studies using non-human primate incisors revealed that the PDL is
regenerated as long as it maintains cellular activity adjacent to the cementum. 19
Therefore a great deal of attention has been paid to the biological storage and
maintenance of PDL cells in these teeth.
Cytokine therapy with growth factors has been suggested as a therapeutic modality
for tissue regeneration due to their capacity to induce a cascade of well-regulated
cellular events involving wound healing.20-22 Basic fibroblast growth factor (bFGF,
FGF-2) is a heparin-binding protein with several physiological functions. It is found in
ectomesenchyme during the embryonal period.23- 25 FGF-2 exhibits potent angiogenic
activities and mitogenic ability of mesenchymal cells within the PDL. 21 Studies have
7
shown that application of FGF-2 enhances the healing of periodontal tissue without
ankylosis, root resorption, or epithelial downgrowth in experimental alveolar bone
defects in beagle dogs21 and primate models.21,26 Seshima et al.27 reported that FGF-2
promotes formation of new PDL and prevents ankylosis and root resorption following
reimplantation of teeth in dogs. Recently, a large-scale multi-center randomized clinical
trial in Japan reported that application of FGF-2 is efficacious in the regeneration of
human periodontal tissue.28 However, the influence of FGF-2 on the periodontal tissue
of transplanted teeth, remains to be elucidated. Given the reported effects of FGF-2 on
periodontal tissue, we hypothesized that healing without root resorption or osseous
repair might be achieved in autotransplantation of tooth with compromised periodontal
ligament, through its effects on angiogenic formation and fibroblastic proliferation.
The objective of this study was to histopathologically investigate the effect of FGF-2
on root resorption following delayed autotransplantation of tooth in dog.
8
Materials and methods
This experiment was performed according to the Guidelines for the Treatment of
Experimental Animals at Tokyo Dental College (No 232202). Fifteen healthy female
beagle dogs (12-month-old, 10-12 kg) were used for this study. Housing and feeding
were according to standard animal care protocols. The animals were under sequential
veterinary control during the entire experimental period.
Surgical procedure
The animals were placed under general anesthesia with sodium pentobarbital
(Somnopentyl®, Kyoritsu Seiyaku, Tokyo, Japan) at a dose of 25 mg/kg. To reduce
stress and hemorrhage in surgical areas, local infiltration anesthesia (2% xylocaine, 1:
80,000 adrenaline) was also used. The second and third mandibular premolars (2P2 and
3P3) were extracted to provide space (recipient sites) for tooth autotransplantation. The
dogs received daily plaque control, consisting of brushing and application of 0.2%
chlorhexidine gluconate solution, in order to establish healthy gingival conditions prior
9
to autotransplantation.
After two months, the tooth transplantation procedure was performed as follows: One
week prior to transplantation, under rubber dam isolation, root canals of each first and
fourth premolar (1P1 and 4P4) were endodontally treated to prevent inflammatory root
resorption from root canal infection. After endodontic access cavities were produced,
the root canals were biomechanically prepared using K- and H-type files. During
instrumentation, the root canals were irrigated with a 10% NaOCl and 3% H2O2
solutions, and later dried with sterile paper points. Then the root canals were filled with
gutta-percha and hydorxyapatite -based root canal sealer (Finapec APC, Kyosera, Kyoto,
Japan), using the lateral condensation method. One week later, a crestal incision was
made from the first to the fourth premolar. Buccal and lingual full thickness flaps were
elevated. The double-rooted 4P4 were separated to allow each root to be tested as an
individual unit. 1P1 and the distal and mesial segments of 4P4 were luxated with an
elevator and extracted with forceps using rotary movements. The roots were then
air-dried on a glass surface for 60 min at room temperature in order to inflict damages
to PDL cells.
10
The recipient sites were prepared using implant drills with copious saline irrigation.
The size and shape of the recipient sites were made as uniform as possible. The sites
were prepared approximately 2 mm wider than the size of the tooth to be transplanted,
according to the method described by Katayama et al.29 After preparation was complete,
recombinant FGF-2 (Lot No. 80HCC; Kaken Pharmaceutical, Tokyo, Japan) was
diluted with distilled water and then mixed with hydroxypropyl cellulose (HPC). The
prepared formulation containing 0.3% FGF-2 (30 µg/site) was applied to the root
surface on the left side (P1 and P4: experimental group) of the mandible. The right side
(1P and 4P: control group) was treated in an identical manner, but without application of
FGF-2 or HPC. HPC was not used in the control group since the application of HPC
alone did not significantly alter the healing of transplanted tooth in our preliminary
experiment.
The teeth (1P1 and 4P4) were placed in the corresponding recipient sites (Fig.1). The
flaps of these recipient sites were then repositioned and sutured. The transplanted roots
were stabilized with a suture splint for 2 weeks. All dogs received antibiotics (Mycillin
Zol KMK, Kawasaki Mitaka Pharmaceutical Co, Kawasaki, Japan; 0.05 ml/kg) for 7
11
days after surgery. They also received daily plaque control regimen consisting of gentle
wiping of the teeth with gauze soaked with 0.2% chlorhexidine gluconate solution. The
sutures were removed after 1 week.
Histological processing
The animals were euthanized with an intravenous overdose of sodium pentobarbital
at 2, 4, or 8 weeks following the procedure described above. The jaw of each animal
was then removed and specimens containing the experimental areas placed in 20%
buffered formalin for 7 days. Specimens were decalcified with 10% ethylenediamine
tetraacetic acid (EDTA) (Wako, Tokyo, Japan) over 5 months, after which they were
dehydrated in ethanol, embedded in paraffin, and serially sectioned to 5 µm thickness in
the buccolingual direction. The sections were then stained with hematoxylin-eosin (H &
E). Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was
performed using an immunoperoxidase staining kit [Histofine SAB-PO (MULTI) Kit;
Nichirei, Tokyo, Japan]. The sections were incubated with mouse anti-PCNA primary
antibody (PC-10; DAKO Corporation, Carpinteria, CA, USA) at a dilution of 1:100.
12
Next, each section was incubated with biotinylated secondary antibody and streptavidin
peroxidase reagents. The presence of peroxidase-complexes was visualized by 3-3’
diaminobenzidine tetrahydrochloride (0.1 mg/ml) solution with 0.65% H2O2. Sections
were counterstained with Mayer’s hematoxylin. A brown coloration indicated a
PCNA-positive reaction. Magnification was set at ×100, and field of connective tissue
in the middle portion of root was selected in each section, which was randomly selected
from each dog. A 0.12-mm2 (0.2 × 0.6 mm) area in the periodontal tissue was submitted
to quantitative analysis as described previously.30
Histomorphometric assessment
All measurements were performed with an image analysis system comprising a
light microscope (Olympus BX51 Microscope, Olympus, Tokyo) with a 4× objective
equipped with a digital camera (HC-2500, Fujifilm, Tokyo). A computer (Precision
Work Station 220 System, Dell, Round Rock, TX, USA) employing an image
processing software (Image Pro Plus v 3.0, Media Cybernetic, Silver Spring, MD, USA)
was used.
13
The analysis of periodontal morphology was a modification of the method described
previously.16, 29 Briefly, the resorption patterns were classified as 1. surface resorption;
small superficial resorption cavities within cementum, 2. dentin resorption; the
condition where bowl-shaped areas of resorption, involving both cementum and dentin;
3. replacement resorption; condition where direct contact between the alveolar bone and
the mineralized root substance (ankylosis) was the characteristic feature.
Three sections (the central part of the tooth and 200-µm sections on either side of that
area) from each transplanted tooth were used for morphometric evaluation. The extent
of each resorption pattern on the root periphery was expressed as a percentage of the
total length of the peripheral contour of the root surface; that is, the distance between
the distal and mesial portions of the cemento-enamel junction. For statistical analysis,
Student’s t test was used to compare differences between experimental and control
groups. P-values less than 0.05 were considered statistically significant.
14
Results
Clinically, the healing response was uneventful in all dogs tested, throughout the
observation periods.
1. Histopathological examination: Week 2
Experimental group:
Newly formed connective tissue covered the roots of tooth transplants (Fig. 2a).
The spaces in the bone and root surface were filled with newly formed connective
tissue composed of spindle-shaped cells, which are associated with growing
capillaries (Fig. 2b). A number of PCNA-positive cells were observed in the newly
formed connective tissue (Fig. 2c). The number of PCNA-positive cells in the
experimental group was statistically significantly greater than that in the control
group (p < .001) (Table I).
Control group:
The root exhibited surface and/ or dentin resorption (Fig. 2d, e). Multinucleated
cells were often observed on the root surface. Few PCNA- positive cells were
observed in the connective tissue (Fig. 2f).
15
2. Histomorphometric assessment: Week 2
The results of histomorphometric assessment performed at 2 weeks after
autotransplantation are presented in Table II. Root resorptions extending to cementum
and/or dentin were already observed in both groups. The occurrence of dentin resorption
in the experimental group was significantly lower than that in the control group (P
< .01). Replacement resorption was not observed in the experimental or control group at
this early stage.
3. Histopathological examination: Week 4 and 8
Experimental group:
At week 4, bone formation had advanced along the root surface (Fig. 3a), and as a
result, PDL-like tissue was observed between the root surfaces and the new bone.
Osteoblast-like and cementoblast-like cells were visible in newly formed bone and
cementum, respectively. It is significant that connective tissue fibers of the newly
formed PDL inserted directly into the newly formed bone.
16
At week 8, the newly formed alveolar bone had advanced to surround the entire root
of the transplanted teeth, and PDL-like tissue was evident between the root surface and
the new bone (Fig. 3b). Cementoblast-like cells were aligned along the surface of the
cementum. In some areas, new cementum had formed on the original cementum. Fibers
had inserted into newly formed cementum and adjacent bone. Only a few cavities
indicating dentin resorption were discernible in the middle and apical portions of the
root. Prevalence of replacement resorption with ankylosis was low.
Control group:
At week 4, areas of root resorption were observed (Fig. 3c). The root surface was
either covered with connective tissue in which collagen fibers ran parallel to the surface
or was the seat of active dentine resorption as evidenced by the presence of
multinucleated cells. Periodontal ligament close to the alveolar bone was partially
occupied by newly formed bone tissue that was in close proximity to or in direct contact
to the dentin surface.
At week 8, the surface of the transplanted root showed signs of replacement
resorption (Fig. 3d). Replacement resorption was observed in the lateral portion of all
17
the roots. Here, resorption was the predominant feature in the middle and apical
portions of the root. In areas with ankylosis, the resorbed dentin surface was in direct
contact with alveolar bone.
4. Histomorphometric assessment: Week 4 and 8
At both observation periods, the occurrence of replacement resorption in the
experimental group was significantly lower than that in the control group (P < .01)
(Table II). No statistically significant difference was found in dentin or surface
resorption between the experimental and control groups.
18
Discussion
In the present study, the root surface treated with FGF-2 achieved a favorable
healing after delayed autotransplantation, whereas the control, non-treated group
exhibited high occurrence of dentin and / or replacement resorption. Uniformity of the
histologic results observed within each group demonstrates that the study design was
sound and is adequate for this type of study. After 4 to 8 weeks, the occurrence of
replacement resorption in the control group was statistically significantly higher than
that in the experimental group. This may be related to the reduction in cellular viability
or activity on the root surface of the transplanted tooth.
The healing process of autotransplantation puts two different tissues in competition:
the PDL on the root surface and the bone tissue of the alveolus.31 In the present study,
the roots were left air-dried for 60 min. prior to transplantation, for the purpose of
damaging the PDL cells. Desiccation for 60 min. or more can cause necrosis of PDL
cells32 and expansion of the injury site in periodontal tissue on the root surface.33 We
speculate that some PDL cells remain vital after 60 min of extra alveolar period, mainly
based on the histopathological findings and data presented by Andreasen11. In a
19
preliminary experiment, we performed toluidine blue staining of the root immediately
after extraction, and observed that most of the PDL tissue appeared to be present on the
root surface. In addition to the reduction of cellular viability by desiccation, it is
possible that damages to PDL and/or cementum by physical stress of extraction are
responsible for the occurrence of surface or dentin resorption at 2 weeks after
transplantation. In competitive wound healing of the damaged or denuded root surface,
repopulation of endosteal osteoblasts would tend to be favored over the limited viable
PDL fibroblasts.22, 34 This may explain high occurrence of replacement resorption
observed in the control group after 4 weeks.
FGF-2 is known to be deeply involved in cell proliferation and differentiation and
also in control of extracellular matrix generation during the processes of tissue
generation and wound healing. 25, 35-39 FGF-2 has been shown to act on a variety of
mesenchymal cell types40.With regard to delayed-replantation of tooth, topical
application of FGF-2 was shown to be effective in prevention of replacement resorption
in animal studies.22, 27 The ability of FGF-2 to stimulate variety of undifferentiated
cell types in periodontal tissue21 lead us to test its potential in enhancing healing of
20
damages induced during the process of autotransplantation. In the present study,
significantly greater numbers of PCNA positive cells were found in the newly formed
connective tissue in the FGF-2 treated group than in the control group. PCNA is a
36-kD polypeptide expressed in the late G1 to S phases of the cell cycle and is a specific
marker for cell proliferation.41 The PCNA positive cells have been shown to be
associated with blood vessels within PDL tissue, and the blastic-cells in the PDL may be
supplied from cells related to these blood vessels.42 Collectively, it was suggested that
cells derived from alveolar side and/or residual PDL of the transplanted tooth with
FGF-2 proliferated in blood clots, reached the surface of the root, and initiated the
formation of an intact PDL. Murakami et al.40, using a canine model, reported that
FGF-2 expression could be detected in immature granulation tissue 1 week after the
operation, and tended to decrease 2 weeks after the operation. The aggregates formed by
FGF-2 are considered to constitute a favorable environment for proliferation of the cells.
Endogenous FGF-2 has been shown to be induced in the early phase of wound healing
in periodontal tissue.40. It is possible that inhibitory effect of FGF-2 on osteoclast-like
cells of monocyte/macrhophage lineage43 played a role in the suppression of dentin
21
resorption in the experimental group at 2 weeks.
At later stages, the FGF-2-treated sites showed formation of intact periodontal tissues
(new cementum, PDL, and supporting bone formation). While the source of PDL
fibroblast-like cells could be, in part, from those viable cells remaining in the PDL
and/or cells on alveolar side, it could be speculated that, under appropriate inductive
stimuli, progenitor cells including paravascular cells could differentiate into a PDL
fibroblastic phenotype22. The high incidence of PDL collagen fibers inserting directly
into newly formed cementum observed in the experimental group after 4W supports this
hypothesis. This finding is consistent with the previous report that the types of cell
which repopulate the root surface determine the nature of attachment during healing.44
FGF-2 downregulates alkaline phosphatase activity and reversibly diminishes the
PDL cells’ potency for mineralized nodule formation21. FGF-2 has been shown to
suppress the expression of bone-related proteins, such as type I collagen, osteocalcin
and bone sialoprotein, but prompted the expression of osteopontin by PDL cells. 45
Furthermore, effects of FGF-2 on hard tissue formation and mineralization may differ
between each cell type, and similarly, functions of oseopontin appear to vary depending
22
on the cell types despite mineralizing and non-mineralizing tissue.46 Taken together, it is
suggested that topical application of FGF-2 stimulates multipotent mesenchymal cells
within the residual PDL22, 26 and/or near alveolar bone, and potentially decreases the
occurrence of replacement resorption, possibly by its differential effects on various
cell-types including those involved in bone remodeling and root resorption.
In the present study, application of FGF-2 did not completely prevent occurrence of
replacement resorption. Since FGFs are structurally and functionally prone to
denaturation under low pH at injury sites46, the myriad biological effects associated with
the exogenous FGF-2 applied in our study might not be sustainable. However, it was
shown that in bone, FGF-2 produced by osteoblastic cells is deposited in bone matrix,
and FGF-2 may be released from the bone matrix and affects bone remodeling.43 The
sustainability of its biological effects may critically influence the manner in which
FGF-2 modulates hard tissue remodeling.
The differences in histomorphometric outcomes between the experimental and
control teeth clearly favor FGF-2 treatment in autotransplantation and could be
considered clinically relevant. Clinical safety of FGF-2 has been shown when applied in
23
conjunction with periodontal surgery: the immunogenic potential of FGF-2 was found
to be extremely low.47 The results from the present study suggest that tissue
engineering to functionally restore the damaged periodontal tissue could ultimately
inhibit replacement resorption.
Acknowledgement
The authors thank Drs. Takashi Inoue, Kan-Ichi Nakagawa, and Kazuyuki Ishihara,
Tokyo Dental College, for helpful discussions.
24
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32
Figure legends
Fig.1
Diagram illustrating transplantation.
33
Fig. 2
Representative photomicrograph of transplanted tooth at week 2 [Experimental (FGF-2
treated) group, a-c; Control group, d-f] Middle portion of the root.
a, Newly formed connective tissue covered root of transplanted tooth. (H&E staining).
b, Spindle-shaped cells and blood vessels adhered to bone surface (H&E).
34
c, A number of PCNA-positive cells (arrows) are observed in the connective tissue
adjacent to the root surface and alveolar bone side (PCNA and counterstaining with
Mayer’s hematoxylin stain).
d, e Root resorption (arrow in d) exdending to dentin area can be observed (H&E)..
f, Only a minimal number of PCNA- positive cells (arrows) are observed in connective
tissue (PCNA and counterstaining with Mayer’s hematoxylin staining).
35
Fig. 3
Repr
[Exp
of th
a, W
had i
b, W
c, We
prese
d, W
resentative p
perimental (F
e root.
Week 4. Bone
inserted into
Week 8. PDL
Week 4. The r
ent (arrows)
Week 8. Bone
photomicro
FGF-2 treat
e formation
o newly form
L-like tissue
root surface
) near the si
e is fused w
graph of tra
ted) group,
is observed
med bone (H
had formed
e exhibits re
ite of resorp
with areas of
36
ansplanted t
a and b; Co
d along the r
H&E staini
d between th
eplacement
ption (H&E)
f root resorp
tooth at wee
ontrol group
root surface
ng).
he root surf
resorption.
).
ption (H&E
ek 4 and we
p, c and d). M
e. Connectiv
face and new
Multinuclea
).
eek 8
Middle port
ve tissue fib
w bone (H&
ated cells ar
tion
bers
&E).
re
37
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