A new method for alveolar bone repair using extracted teeth for the graft material.

Abstract

In the clinical field of jawbone formation, the use of autogenous bone as the graft material is the gold standard. However, there are some problems with this technique, such as risk of infection on the donor side, the limited amount of available bone mass, and marked resorption of the grafted bone. We investigated the potential for using teeth as a bone graft material for jawbone formation because the dental pulp contains stem cells, including undifferentiated neural crest-derived cells.
Alveolar bone defects were created in Wistar rats, and the defects were filled with either tooth or iliac bone graft material, or left as controls. The potential for using teeth as a bone graft material for jawbone formation was measured using real-time polymerase chain reaction, microcomputed tomography, and histologic analysis.
Polymerase chain reaction revealed that the expressions of P75, P0, nestin, and musashi-1 were significantly higher in teeth than in mandibular bone and iliac bone grafts. Hematoxylin and eosin staining and microcomputed tomography showed that at 8 weeks, tooth graft material produced a similar amount of new bone compared to iliac bone graft material. Osteopontin was expressed in both the tooth and iliac bone graft material at 6 and 8 weeks after surgery. Dentin sialoprotein was expressed in the tooth graft material in the new bone at 6 weeks only.
These results indicate that teeth may be an alternative material to autogenous bone for treating alveolar bone defects by grafting.

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A New Method for Alveolar Bone Repair
Using Extracted Teeth for the Graft
Material
Tomoki Nampo,* Junichi Watahiki,* Akiko Enomoto,* Tomohiro Taguchi,* Miki Ono,*
Haruhisa Nakano,* Gou Yamamoto,†Tarou Irie,†Tetsuhiko Tachikawa,†and Koutaro Maki†
Background: In the clinical field of jawbone formation, the
use of autogenous bone as the graft material is the gold stan-
dard. However, there are some problems with this technique,
such as risk of infection on the donor side, the limited amount
of available bone mass, and marked resorption of the grafted
bone. We investigated the potential for using teeth as a bone
graft material for jawbone formation because the dental pulp
contains stem cells, including undifferentiated neural crest–
derived cells.
Methods: Alveolar bone defects werecreatedinWistar rats,
and the defects were filled with either tooth or iliac bone graft
material, or left as controls. The potential for using teeth as
a bone graft material for jawbone formation was measured
using real-time polymerase chain reaction, microcomputed
tomography, and histologic analysis.
Results: Polymerase chain reaction revealed that the ex-
pressions of P75, P0, nestin, and musashi-1 were significantly
higher in teeth than in mandibular bone and iliac bone grafts.
Hematoxylin and eosin staining and microcomputed tomog-
raphy showed that at 8 weeks, tooth graft material produced
asimilaramountofnewbonecomparedtoiliacbonegraftma-
terial. Osteopontin was expressed in both the tooth and iliac
bone graft material at 6 and 8 weeks after surgery. Dentin sia-
loprotein was expressed in the tooth graft material in the new
bone at 6 weeks only.
Conclusion: These results indicate that teeth may be an al-
ternative material to autogenous bone for treating alveolar
bone defects by grafting. J Periodontol 2010;81:1264-1272.
KEY WORDS
Bone regeneration; bone substitute; grafts, bone; neural
crest; tooth.
I
(e.g., demineralized freeze-dried bone
allografts and freeze-dried bone allo-
grafts); xenografts, (e.g., bovine bone
and coral); and alloplasts, (e.g., ce-
ramics for biologic use, b-tricalcium
phosphate [b-TCP] and hydroxyapatite).
Three properties are required for an ideal
bone graft material: 1) osteoconduction,
which provides scaffolds for bone regen-
eration;12) osteoinduction, which pro-
motes the recruitment of bone-forming
cells, such as undifferentiated cells and
preosteoblasts, and formation of bone
from these cells;1,2and 3) osteoprolifer-
ation, the induction of cells contained in
the graft material to promote bone re-
generation.3Allografts lack osteoprolif-
eration, and xenografts and alloplasts
only show osteoconduction. Because
only autogenous bone exhibits all three
properties, autogenous bone grafting is
currently considered the best method.4
The iliac bone is a frequently used auto-
genous bone and it is grafted into alve-
olar bone defects in most cases of cleft
palate.5However, there are problems
with autogenous bone grafting, such as
risk of infection at the donor side, limited
amount of available bone mass, and
marked resorption of the grafted bone.6,7
Developmentally, most bones of the
trunk and extremities, including the
iliac bone, are formed by endochondral
n the field of clinical dental bone
formation, various bone graft mate-
rials are used. These include allografts
* Department of Orthodontics, School of Dentistry, Showa University, Tokyo, Japan.
† Department of Oral Pathology and Diagnosis, School of Dentistry, Showa University.
doi: 10.1902/jop.2010.100016
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ossification, whereas the jaw and alveolar bones are
formed by intramembranous ossification.8Using
bone with a different ossification pattern from those
ofthejawandalveolarbonesasagraftmaterialforjaw-
bone reconstruction is a matter of concern. Donovan
etal.9andDonosetal.10graftediliacandcranialbones
and investigated the graft bone resorption rate after 6
months.Theyobservedthat about twice asmuchiliac
bone was resorbed compared with grafted cranial
bone. Jaw and alveolar bonesbothdifferentiatefrom
neural crest cells.
Vertebrates develop from three germ layers, ecto-
derm, mesoderm, and endoderm, and a type of tis-
sue originating from neural tube fusion region, neural
crestcells.Thisiscalledthefourthgermlayerbecause
of its importance. Neural crest–derived cells have
been shown to exhibit multipotency and differentiate
into mesodermal mesenchymal cells, despite being
ectodermal.Theyshowahighcapacityforself-regen-
eration and persist in adult tissues.11,12
Tissues derived from the neural crest include the
maxillofacial bones (excluding the occipital, sphe-
noid, temporal, and ethmoid bones); cartilage; teeth;
and nerve and glial cells.13,14Of these, teeth contain
stem cells in the dental pulp, and it has been sug-
gested that the dental pulp contains undifferentiated
neural crest–derived cells.15-17Seo et al.18cultured
stem cellsisolated from dental pulp, grafted them into
a defect prepared in the cranial bone, and observed
hard tissue formation. In addition, dentin contains
growth factors: insulin-like growth factor (IGF)-II,
bonemorphogeneticprotein(BMP)-2,andtransform-
ing growth factor (TGF)-b.19Cementum contains
TGF-b, IGF-I, and type I and III collagen.20Saygin
et al.21suggested that the use of cementoblasts for
periodontal tissue regeneration is worthwhile. Isaka
et al.22reported that the periodontal ligament has
the ability to regenerate bone, and Flores et al.23re-
generated periodontal tissue using cultured peri-
odontal ligament cells. The periodontal ligament
also contains TGF-b, IGF-I, basic fibroblast growth
factor, vascular endothelial growth factor, BMP-2,
platelet-derived growth factor (PDGF), and type I col-
lagen.24Furthermore, dentin and cementum contain
proteins common to bone, such as osteopontin
(OPN), bone sialoprotein (BSP), osteocalcin, dentin
sialoprotein (DSP), dentin matrix protein-1 (DMP-1),
type I collagen, osterix, and Cbfa1 (Runx2). These
are reportedly involved in bone formation and resorp-
tion.25,26
Thus, we considered that teeth containing undiffer-
entiated neural crest–derived cells, proteins involved
in bone formation, and growth factors may be used
as a bone graft material for jawbone formation.
Although there has been no study in which teeth
were usedasabone graftmaterial, tooth replacement
bybonehasbeenreported.Inthispreviouscase,os-
teoclast cells appeared in the pulp cavity after tooth
reimplantation and the pulp was replaced by bone
tissue, followed by root resorption and ankylosis. Fi-
nally, the whole root was integrated into the sur-
rounding alveolar bone.27-29These reports show
that the jawbone and teeth have a high level of affinity
for each other.
Basedonthepreviousinformation,weinvestigated
the possibility of using teeth as a bone graft material
for jawbone formation by comparing it to autoge-
nous iliac bone grafts.
MATERIALS AND METHODS
Real-Time Polymerase Chain Reaction (PCR)
RNA isolation. Tooth, iliac bone, and mandibular
bone (control) were extirpated from 12-week-old
male Wistar rats (350 to 400 g), and RNA was ex-
tracted from the tissue exposed to the RNA stabi-
lizing treatment‡according to the manufacturer’s
protocol.
Reverse transcription. After the isolation of the
RNA, a reverse transcriptase (RT) kit§was used to
make cDNA. The mixture was composed of 13 ml of
total RNA, 2 ml of random primers, 2 ml of deoxynu-
cleotide triphosphate, 2 ml of buffer RT, and 1 ml of
omniscript RT added to a final volume of 20 ml. The
mixture was heatedifor 60 minutes at 37?C.
Semiquantitative PCR. The cDNA from the RT re-
action was used as a template in the PCR. We used
four primers (P75,¶P0,#nestin,** and musashi-1††).
The PCR mixture was composed of 2 ml of sample
cDNA, 1 ml of each primer, 7 ml of RNase-free water,
and 10 ml of a gene expression mix‡‡for to a final vol-
ume of 20 ml. We performed real-time PCR§§for 50
cycles at 95?C for 15 seconds, 60?C for 1 minute fol-
lowedby50?Cfor2minutes,and95?Cfor10minutes.
Wecalculatedtherelativeexpressionlevelbydividing
the signal intensity of each gene by that of GAPDH.ii
For quantification, a series of five-fold dilution stan-
dards and a negative control (RNase-free water) were
run alongside the samples.¶¶
Animals
Sixty 12-week-old male Wistar rats were randomly di-
vided into three groups of 20. Ten rats in each group
were sacrificed at 6 weeks, and the remaining 10 at 8
weeks. The groups were as follows: group 1 (tooth
‡
§
i
¶
#
** Rn00564394_m1, Applied Biosystems.
†† Rn00596059_m1, Applied Biosystems.
‡‡ TaqMan Gene Expression Master Mix, Applied Biosystems.
§§ 7500 ABI PRISM, Applied Biosystems.
ii
Rn99999916_s1, Applied Biosystems.
¶¶ Applied Biosystems.
RNAlater, Qiagen, Hilden, Germany.
Omniscript Reverse Transcriptase Kit, Qiagen.
2400 GeneAmp PCR System, PerkinElmer Japan, Tokyo, Japan.
Rn00586061_s1, Applied Biosystems, Foster City, CA.
Rn00566746_m1, Applied Biosystems.
J Periodontol • September 2010
Nampo, Watahiki, Enomoto, et al.
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Page 3

group), tooth except enamel with b-TCP##complex;
group 2 (bone group), iliac bone with b-TCP complex
(positive control); and group 3 (control group), no ma-
terial (negative control). The Animal Research Com-
mittee of Showa University, Tokyo, Japan, approved
all procedures.
Graft Material Preparation
Allsurgicalprocedureswereperformedundergeneral
anesthesia insterile conditions. Afterinhalation ofan-
esthesia with ethyl ether,*** general anesthesia was
achievedwithanintraperitonealinjectionofpentobar-
bital sodium.†††In the tooth group, a tooth was ex-
tracted on the side opposite to the area where the
alveolar bone defect was made. The crown portions
of the extracted teeth were removed with scissors,
and the root portions of the remaining teeth were
trimmed as closely as possible to 500 mm at once.
Next, the trimmed tooth was mixed with a measured
quantity of b-TCP. These grafts were prepared on ice.
In the bone groups, the cancellous bone of the iliac
bone was removed, granulated, and mixed with
b-TCP. The graft material used in both groups was
approximately 0.2 g. The ratio of the mixture of the
transplant material and b-TCP was adjusted to 2:1.
Surgical Protocol
Anincisionwasmadeinthepalate,andafull-thickness
flap was created exposing the alveolar bone. An alve-
olar bone defect, 2 mm in diameter, was made with
a diamond bur. Subsequently, one of the two mate-
rials was grafted into each alveolar bone defect (the
control group did not receive implantation of a graft
material), and a resorbable bilayer collagen mem-
brane‡‡‡was placed over the bone defect in all the
groups. All the graft materials were transplanted to
thedefectwithin30minutesafterextirpation.Theflap
was repositioned and sutured tightly with resorbable
sutures,§§§covering the bone defect.
Post-surgical Care
All the rats received antibiotics (penicillin G potas-
sium, 200,000 unitsiii) intramuscularly daily for 3
days after surgery. The rats were fed a soft diet¶¶¶
for 2 weeks to reduce any potential mechanical dam-
age.
Microcomputed Tomography (m-CT)
Observations
m-CT###wasusedtoobservenewboneformation.Im-
ageswereacquiredimmediatelyaftersurgeryandat6
and 8 weeks. We confirmed the maxillary-bone form
of an intact rat by a pilot experiment.
Histologic Procedures
Animals were euthanized 6 and 8 weeks after surgery
with an overdose of ethyl ether. All defects in the
groupsweredissected along with thesurroundingsoft
and hard tissues. Block sections were fixed with 4%
paraformaldehyde, decalcified**** for 2 days, neu-
tralized with 5% sodium sulfate anhydrous, and then
embedded in paraffin. Sections were cut, deparaffi-
nized, and stained with hematoxylin and eosin
(H&E). We confirmed the maxillary-bone form of an
intact rat by a pilot experiment in the tissue section.
Immunohistochemical Procedures
Sections (7 mm) were cut, mounted on slides, depar-
affinized, and incubated with anti-OPN antibody††††
and anti-DSP antibody‡‡‡‡at appropriate dilutions.
Specimens to be reacted with anti-DSP antibody were
pretreated by using the activator§§§§for 10 minutes.
RESULTS
Real-Time PCR
Real-time PCR results for the expressions of P75, P0,
nestin, and musashi-1 in the tooth, iliac bone, and
mandibular bone (control) were quantitatively deter-
mined (Fig. 1). The expressions of all four genes were
significantly higher in the tooth than in the mandibu-
larboneandiliacbonegraftmaterial.Intheiliacbone,
the expressions of all four genes were insignificant.
Clinical Observations
Wound healing was uneventful. Six weeks after sur-
gery,thewoundsofthetooth,bone,andcontrolgroups
appeared to be similar. No material exposure or in-
tense inflammatory reactions were observed during
the healing period.
m-CT Observations
The absorption of grafted materials and the formation
of new bone with time in both the tooth and bone
groups were confirmed with m-CT (Figs. 2 and 3). In
the bone group, new bone formation exceeding the
defective bone region was noted 6 weeks after sur-
gery, but markedresorption of thenew bone occurred
at 8 weeks. In the tooth group, new bone formation
filled the bone defect at 6 weeks, and the new bone
was retained at 8 weeks. Little new bone formation
was noted in the control group at 8 weeks.
Histologic Observations
In the tooth group, slight inflammatory reactions were
notedaroundthedefectsat6weeks,butnewbonefor-
mation was confirmed in the defects. In the bone
##
***
†††
‡‡‡
§§§
iii
¶¶¶
###
Curasan, Kleinostheim, Germany.
Wako Pure Chemical Industries, Osaka, Japan.
Kyoritsu Seiyaku Corporation, Tokyo, Japan.
Geistlich Pharma.
BEAR Medic Corporation, Ibaraki, Japan.
Meiji Seika Kaisha, Tokyo, Japan.
Nihon Nosan Kogyo Corporation, Yokohama, Japan.
eXplore Locus CT System and MicroView, GE Healthcare, Tokyo,
Japan.
**** KALKITOX, Wako Pure Chemical Industries.
†††† Cosmo Bio, Tokyo, Japan.
‡‡‡‡ Santa Cruz Biotechnology, Santa Cruz, CA.
§§§§ Gold Standard Series L.A.B. Solution, Polysciences, Warrington, PA.
Bone Graft Material Made From Extracted Teeth
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group, slight inflammatory re-
actions were similarly shown.
The volume of new bone for-
mation exceeded the defec-
tive bone region at 6 weeks.
Furthermore, a large amount
of bone marrow was formed
compared to the tooth group.
In the control group, little new
bone was formed at either 6
or 8 weeks. No signs of in-
flammation were observed in
any defects 8 weeks after sur-
gery. New bone resorption at
8 weeks was marked com-
pared to that at 6 weeks in the
bone group. In contrast, new
bone mostly filled the defects
at 8 weeks in the tooth group
(Fig. 4).
Immunohistochemical
Observations
In the surroundings of the newly
formed bone in both the tooth
and bone groups at 6 and 8
weeks, OPN was more widely
expressed. In the control group,
there was little expression of
OPN at both 6 and 8 weeks.
DSP was expressed in the tooth frag-
ment graft. In the bone defect, the ex-
pression was positive at 6 weeks, but
hardly expressed at 8 weeks (Figs. 5
and 6).
DISCUSSION
We investigated the usefulness of teeth
asa bone graft material for jawbone for-
mation by comparing grafts done with
teeth and autogenous iliac bone.
The treatment of defects made in the
cranial bone of rats using graft mate-
rials has been reported.18,30However,
it has been reported that some parts of
the cranial bone are derived from de-
velopmentally different cells. Yoshida
et al.13reported that the frontal bone is
derived from neural crest cells, whereas
the temporal bone is derived from the
mesoderm. In accordance with Park
etal.,31weusedthejawboneforthegraft
bed.
We determined the evaluation times
for m-CT and histologic examination at
6 and 8 weeks after grafting based on
Figure 1.
Expressions of P75 (A), P0 (B), Nestin (C), and Musashi-1 (D) in tooth, iliac bone, and ungrafted
control(mandibularbone).Twenty12-week-oldmaleWistarratswererandomlydividedintofourgroupsof
five rats each. All the expressions of four genes were significantly higher in tooth than in mandibular bone
and iliac bone.
Figure 2.
m-CTslices(80kV/450mA,93-mmslicethickness)andthree-dimensionalimagesofthebone
defect (2-mm diameter, 2-mm depth). Purple dashed line = part of the graft; yellow solid
line = edge of the bone defect; blue arrowheads = graft material.
J Periodontol • September 2010
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Page 5

the healing process of rat jawbone tissue, as reported
by Schmitz et al.32and Hyun et al.33
Shapoff et al.34reported that the particle size of
graftmaterialsinfluencedlaterboneformation.Bhaskar
et al.35reported that the ideal particle size of bone graft
materials is 500 mm and that the between-particle dis-
tance is 150 mm. These sizes were recommended
because resorption requires a prolonged time if the
particle size is too large, and particles are resorbed
before they are able to function as a graft material if
the size is too small. The retention of blood clots is dif-
ficult when the between-particle distance is too large,
whereas blood vessels cannot readily enter the mate-
rial when the distance is too small.36Because it was
difficult to prepare particles with a homogeneous size
from autogenous tissues, we added b-TCP to reduce
the difference in the between-particle distance be-
tween the iliac bone grafts and extracted tooth grafts.
Enamel, an epithelial tissue, was completely re-
moved from the teeth used for bone graft material.
The remainder of the teeth including the dentin, ce-
mentum, pulp, and periodontal ligament were ground
and immediately used for grafting.
GeneexpressionsofP75,P0,nestin,and musashi-1
were significantly higher in the tooth group than in
mandibular and iliac bone groups using real-time
PCR.P75andP0havebeenrecentlydescribedasneu-
ral crest cell markers. P75 is the founding member
of the tumor necrosis factor receptor superfamily.
This family of receptors is distinguished by multiple
cysteine-richdomainsforligandbinding,asingletrans-
membrane sequence, and a non-catalytic cytoplasmic
Figure 3.
m-CTimagesshowingthetoothgraftsimmediatelyaftersurgery(A),6weeksaftersurgery(B),and8weeksaftersurgery(C).m-CTimageshowingtheiliac
bonegraftsimmediatelyaftersurgery(D),6weeksaftersurgery(E),and8weeksaftersurgery(F).m-CTimageincontrolgroup(nograft)immediatelyafter
surgery (G), 6 weeks after surgery (H), and 8 weeks after surgery (I).
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