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British Journal of Oral and Maxillofacial Surgery 46 (2008) 110–113

Processed bovine dentine as a bone substitute

Keyvan Moharamzadeh a,∗ , Christine Freeman b,1 , Keith Blackwood a
aSchool of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield S10 2TA, UK
bSheffield Teaching Hospitals NHS Trust, Charles Clifford Dental Hospital,
Wellesley Road, Sheffield S10 2SZ, UK

Accepted 25 July 2007

Available online 25 September 2007

Abstract

Objectives: Different forms of allogenic dentine have been studied for their potential use as bone substitutes. We report a new method for
processing bovine dentine that results in a sterile bioactive material for repair and regeneration of bone.
Methods: Extracted bovine dentine was processed mechanically and chemically with inorganic and organic solvents, and sterilised. The in
vitro biocompatibility on human gingival fibroblasts was assessed by the Alamar Blue assay and the in vivo biocompatibility evaluated by
implantation of the processed dentine into rats’ femurs.
Results: The dentine showed excellent biocompatibility in vitro, stimulated formation of new bone and was completely incorporated into the
new bone in vivo.
Significance: Processed bovine dentine has the potential to be used as a suitable substitute in bone repair and regeneration.
© 2007 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Keywords: Dentine; Bone regeneration; Biocompatibility; Alamar Blue assay

Introduction dentine is non-irritant and compatible with periapical tissue

Substitutes for bone provide osteoconductive scaffolding
similar to those of autogenous bone. They eliminate mor-
bidity of the donor site; reduce operating time, complexity,
and treatment costs; and improve satisfaction for patients.1
Replantation of avulsed teeth after elimination of the peri-
odontal ligament results in dentoalveolar ankylosis, followed
by long-term resorption of roots and replacement of bone.2
This phenomenon indicates that dentine has the potential to
be used as a suitable osteoconductive material in repair and
regeneration of bone. Different forms of allogenic dentine
have been studied for their potential use as bony substitutes.
These include freeze-dried dentine, demineralised dentine,
and particulate dentine (tooth ash). Freeze-dried allogenic
∗
Corresponding author. Tel.: +44 114 2717924; fax: +44 114 2665326.
E-mail addresses: k.moharamzadeh@sheffield.ac.uk
(K. Moharamzadeh),c.o.freeman@sheffield.ac.uk (C. Freeman),
K.Blackwood@sheffield.ac.uk (K. Blackwood).
1 Tel.: +44 114 2717849; fax: +44 114 2717863.
as an apical barrier material;3 it has also been used clini-
cally in the repair of periodontal osseous defects.4 It has been
reported that demineralised dentine stimulates new bone for-
mation and completely incorporates into the new bone. It is
then resorbed during repair of bone and results in fast heal-
ing of bony defects.5,6 Particulate dentine has been studied
in its pure form or combined with Plaster of Paris as a bony
substitute. However, these materials are less effective than
processed bone-derived products.7
To obtain a pure dentine matrix and eliminate all other
components that might cause inflammatory reactions, we
have developed a new method for processing bovine den-
tine that results in a sterile bioactive material for bony repair
and regeneration in periodontal, maxillofacial, or other bone
defects.
The aim of this study was to assess in vitro and in vivo
biocompatibility of the processed dentine and to evaluate its
effectiveness in repair and regeneration of bone.

0266-4356/$ – see front matter © 2007 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2007.07.209
 K. Moharamzadeh et al. / British Journal of Oral and Maxillofacial Surgery 46 (2008) 110–113 111

Material and methods sutures and the animals allowed to recover. They were main-
tained for 4 weeks and then killed by cervical dislocation
Processing of dentine (schedule one method). The right femurs were dissected free
and placed in formalin before decalcification and processing
Extracted young bovine teeth with open apices were washed to haematoxylin and eosin stained paraffin sections.
with water, cleaned, and all soft tissues including the pulp and
periodontal ligament were removed from the root. After the
enamel had been removed with a high-speed diamond bur, Results
the dentine was broken into small pieces (5–10 mm) using
a mortar and pestle. After boiling the dentine fragments in In vitro test
distilled water for 2 h, they were refluxed in isopropanol for
During incubation, the colour of Alamar Blue changed from
2 h to remove any remaining soft tissue or fat. After they
blue to red. The presence of processed dentine did not inhibit
had been washed with distilled boiling water several times to
the chemical reduction of Alamar Blue. In fact, statistical
eliminate the organic solvent, the fragments were air dried at
analysis by one-way ANOVA showed that there was an
100 ◦ C. Final stages involved grinding pieces of dentine into
increase in the cell viability of the test group, mean (SD)
small particle powder using a high-speed mechanical blender,
being 284.1 (2.9) compared with 274.9 (7.0).
packaging, and sterilisation by ␥-irradiation.

In vitro biocompatibility test In vivo test

Primary human gingival fibroblasts were obtained from Histological examination of the implanted dentine showed
stocks stored in liquid nitrogen. These cells had already complete incorporation of the particles of dentine into newly
been harvested from biopsy specimens from patients with formed bone (Fig. 1). There were no signs of chronic inflam-
Ethics Committee approval. The cells were cultured in mation, giant cells, or foreign body reactions to the implant
Dulbecco’s Modified Eagle Medium (Sigma, UK) supple- (Fig. 2). Numerous osteoblasts were found on the periphery
mented with 10% fetal calf serum (Biowest Ltd., UK), of the newly formed bone around the dentine. In some areas
l-glutamine (Sigma, UK) 2 mM, penicillin 100 U/ml (Sigma, dentine had been resorbed by the osteoclasts and replaced by
UK), and streptomycin 100 ␮g/ml (Sigma, UK). Cultures new bone (Fig. 3).
were incubated at 37 ◦ C in a humidified atmosphere of 5%
carbon dioxide in air. In vitro biocompatibility of the graft
material was assessed by culturing human gingival fibrob- Discussion
lasts in the vicinity of the powder in cell culture inserts.
100,000 cells/well were seeded into 24-well plates, cultured Bovine dentine was successfully processed and sterilised in
for 24 h, and then exposed to dentine powder in cell cul- this study for the in vivo use as a bone substitute. By boiling
ture inserts for 72 h (n = 4). Cells with empty inserts were
used as controls. Cell viability was assessed by Alamar Blue
assay. After the incubation period the inserts and the medium
were removed and solution of 10% Alamar Blue (Biosource,
Camarilo, CA) in 1 ml Dulbecco’s Modified Eagles Medium
(DMEM) was added to each well. After 5 h incubation, 200 ␮l
of each well were placed into 96-well plate in duplicates, and
the fluorescence intensity was measured using a fluorescent
plate reader (FLUOstar Galaxy, BMG Lab technologies) at
an excitation wavelength of 530 nm and emission wavelength
of 580 nm. Data were analysed by one-way ANOVA.

In vivo biocompatibility test

Six male rats aged 4–5 weeks, were used in the study. They
were housed together under standard laboratory conditions
and had free access to food and water. Samples of sterile
dentine were placed into a defect created in the mid-shaft of
the right femur under aseptic conditions. They were oper-
ated on under general anaesthesia, and a single bone defect Fig. 1. Histological examination of dentine implanted into the bone, showing
was created in the femur using a stainless steel dental bur with complete incorporation of the dentine particle into the newly formed bone
sterile saline irrigation; wounds were closed using resorbable (haematoxylin and eosin, original magnification ×40).
112 K. Moharamzadeh et al. / British Journal of Oral and Maxillofacial Surgery 46 (2008) 110–113
Fig. 2. The bone marrow is normal in appearance with no evidence of
a foreign body response to the dentine (haematoxylin and eosin, original
magnification ×10).
the dentine fragments in distilled water followed by refluxing
in isopropanol, we removed the antigenic materials com-
pletely from the dentine, leaving the mineral structure and
inert organic matrix.
We used the Alamar Blue assay for the assess-
ment of its biocompatibility. This assay incorporates an
oxidation–reduction indicator that both fluoresces and
changes colour in response to chemical reduction of growth
medium that results from cell growth.8 Alamar Blue has two
advantages over the commonly used MTT assay; firstly its
change in colour can be detected both spectrophotometri-
cally and fluorometrically, which gives greater sensitivity of
detection. Secondly, as it is not toxic to cells it is possible
to assess their viability on more than one occasion.9 Our
results showed that the processed dentine was not only bio-
compatible in vitro, but also stimulated the proliferation of the
Fig. 3. Dentine resorption and subsequently deposition of new bone by the
osteoblasts (haematoxylin and eosin, original magnification ×20).
fibroblasts and increased cell viability. To our knowledge this
finding has not been previously reported, and suggests that the
processed dentine particles can promote cell proliferation. It
also indicates that the method described for the processing of
the dentine removes the remnants of fat and cells efficiently
without leaving any toxic organic solvent inside the dentinal
tubules.
In vivo implantation testing of the dentine also confirmed
the results of the in vitro test. Good biocompatibility with-
out eliciting inflammatory reaction or infiltration of foreign
body giant cells and the ability to be replaced by the new
bone, indicate that the processed dentine has the poten-
tial for development as an osteoconductive bone substitute.
Osteoconduction is characterised as bony growth by appo-
sition from the surrounding bone. This process provides a
physical matrix or scaffolding suitable for the deposition of
new bone.10 Materials commonly used for osteoconductive
bone grafts are alloplasts such as bioactive ceramics and
xenografts.11–13 The advantage of dentine over other bone
substitutes such as hydroxyapatite is that in addition to the
mineral component, dentine contains more than 20% organic
matrix similar to that of bone.
On the basis of our limited results the material is biocom-
patible in bone for the duration of the study. Further long-term
comparative studies are under way to develop this material
further as a potential substitute for bone.
Acknowledgements
We thank Professors Ian Brook and Richard Van Noort for
their financial support and guidance.
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