Archives of Orofacial Sciences 2006; 1: 15-20
ORIGINAL ARTICLE
Mitotic Index and Chromosomal Analyses for Hydroxyapatite
Implantation in Rabbits
T.P. Kannan1*, B.B. Quah2, A. Azlina1, A.R. Samsudin1
1
School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; 2 Klinik Pergigian,
Sungai Bakap, 14200 Pulau Pinang, Malaysia
*Corresponding author: tpkannan@kb.usm.my
ABSTRACT
Dentistry has searched for an ideal material to place in osseous defects for many years. Endogenous bone replacement
has been the golden standard but involves additional surgery and may be available in limited quantities. Also, the
exogenous bone replacement poses a risk of viral or bacterial transmission and the human body may even reject them.
Therefore, before new biomaterials are approved for medical use, mutagenesis systems to exclude cytotoxic,
mutagenic or carcinogenic properties are applied worldwide. The present preliminary study was carried out in five
male New Zealand white rabbits (Oryctolagus cuniculus). Porous form of synthetic hydroxyapatite granules (500 mg),
manufactured by School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Penang, was
implanted in the femur of the rabbits. Blood samples were collected prior to implantation and one week after
implantation. The blood was cultured in vitro and the cell division was arrested at metaphase using colcemid. This was
followed by the hypotonic treatment and fixation. Then, the chromosomes were prepared and stained for analysis. The
modal chromosome number of rabbit (Oryctolagus cuniculus) was found to be 2n=44. The mean mitotic index values
prior to and after implantation were 3.30 ± 0.66 and 3.24 ± 0.27 per cent respectively. No gross chromosome
aberrations, both numerical and structural were noticed either prior to or after implantation of the biomaterial. These
findings indicate that the test substance, synthetic hydroxyapatite granules does not produce gross chromosome
aberrations under the present test conditions in rabbits.
Key words: hydroxyapatite, mitotic index, chromosome aberration
_______________________________________________________________________________
However, synthetic hydroxyapatite is
relatively a new biomaterial and before new
materials are approved for medical use,
mutagenesis systems to exclude cytotoxic,
mutagenic or carcinogenic properties are applied
worldwide (Katzer et al., 2002). The
chromosome aberration test using cultured
mammalian cells is one of the sensitive methods
to predict mutagens and/or carcinogens and is a
complementary test to the Salmonella/microsome
assay (Ishidate et al., 1998). This study aims to
find a possible chromosomal effect induced by
the synthetic porous hydroxyapatite granules
(Manufactured by School of Materials and
Mineral Resources Engineering, Universiti Sains
Malaysia, Penang) in the lymphocytes of rabbits.
INTRODUCTION
There is a necessity for replacing bone substance,
which has been lost due to traumatic or nontraumatic events. The lost bone can be replaced
by endogenous or exogenous bone tissues. For
these reasons, there is a growing need for
fabrication of artificial hard tissue replacement
implants and biomaterials may provide a solution
for these problems. An ideal bone substitute
should be biocompatible, which means
acceptance of the implant to the tissue surface
(Wojciech and Masahiro, 1998).
Hydroxyapatite [Ca10(PO4)6 (OH)2] is the
biomaterial of choice in the present study.
Hydroxyapatite is a main mineral compound of
calcium phosphate in bone and teeth and in
addition to being biocompatible, hydroxyapatite
has also been shown to possess osteoconductive
properties (Bauer et al,.1991; Flatley, 1983;
Sartorius, 1986). Synthetic hydroxyapatite has
been evaluated for use in ridge augmentation,
orthognathic reconstruction and periodontics.
MATERIALS AND METHODS
Ethical approval
Approval for this study was obtained from the
Animal Ethics Committee, Universiti Sains
Malaysia, Health Campus, Kubang Kerian,
Kelantan as per the reference. PPSG/07(A)/044
dated 28.3.2004. All procedures including animal
handling in this study complied with the Animal
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Kannan et al.
Ethics Guidelines as formulated by the Animal
Ethics Committee, Universiti Sains Malaysia.
Post-implantation blood collection
The second blood collection was performed
seven days after implantation. 0.3 ml of blood
was collected from the ear vein by aseptic
techniques using a tuberculin syringe and
immediately transferred to sterile heparinised
vial.
Animals
A total of five healthy adult male New Zealand
White rabbits (Oryctolagus cuniculus), of 4 to 5
months of age were used in the study. The mean
body weight of the animals was 2.64 ± 0.05 kg.
Each rabbit was reared in separate cage and were
fed ad libitum with standard rabbit pellets
manufactured by Gold Coin Feed Mills Sdn.
BhD, Malaysia and water was made available
throughout.
Lymphocyte
culture
for
metaphase chromosomes
obtaining
The cultures were set up in disposable 15 ml
centrifuge tubes using 8 ml of RPMI 1640
medium, 0.1 ml (100 µg) of phytohaemagglutinin
M, 1.5 ml of fetal bovine serum, 0.1 ml of
antibiotic solution (Penicillin G Sodium – 5000
units and Streptomycin Sulphate - 5000 µg) and
0.3 ml of whole blood so that the total volume of
culture was 10 ml. The contents were mixed well
by gentle shaking of the tubes and incubated at 37
˚C for 72 hours (Fig. 2). The cultures were gently
agitated twice a day manually, which increased
the number of mitoses (Halnan, 1977). The cell
division was arrested at metaphase by adding 0.1
ml (1 µg) of colcemid to the cultures 1½ hours
prior to harvesting. This was followed by
hypotonic treatment using pre-warmed potassium
chloride solution (0.56 per cent) for 30 minutes
and fixation using 3:1 Methanol:Acetic acid. The
cells were centrifuged at 1000 rpm for 10 minutes
throughout to sediment the cells.
Blood collection
Collection of blood sample was performed twice,
once prior to the implantation of the biomaterial,
and another post-implantation. 0.3 ml of blood
was collected from the ear vein by aseptic
techniques using a tuberculin syringe and
immediately transferred to sterile heparinised vial
prior to implantation of the biomaterial.
Control and test samples
The blood collected and analyzed prior to
implantation of biomaterial acted as the control
and those collected after the implantation served
as the treatment samples.
Anaesthesia
The rabbits were restrained using the standard
rabbit restrainer and anaesthetized using xylazine
(5 mg/kg body weight) and ketamine (35 mg/kg
body weight) intra muscularly.
Preparation of metaphase chromosome spread
Chromosome spreads were prepared from each
rabbit sample collected prior to and after the
implantation by gently dropping the cell
suspension from a height of 1 foot on to a clean
grease free slide and air-dried. The slides were
stained with 10 per cent Leishman’s stain in
phosphate buffer saline (pH 6.8) for 5 minutes.
The slides were dried, mounted and the
chromosomes were observed under the
microscope.
Biomaterial
The biomaterial used in this study is a porous
form of synthetic hydroxyapatite granules, which
is manufactured by the School of Materials and
Mineral Resources Engineering, Universiti Sains
Malaysia, Penang, Malaysia. The granules ranged
from 100 to 200 microns in size.
Implantation of biomaterial
The skin and muscles were incised and a cavity
(2.5cm x 0.5cm x 0.5cm) was made using a
surgical bur in the right femur of each rabbit.
Exactly 500 mg of the biomaterial was then
implanted (Fig. 1) using a spatula. Then, the
muscles and skin were sutured. This whole
procedure took approximately 1½ hours and
utmost care was taken to see that the procedure
was done in a sterile manner. The animal was
given antibiotic coverage for 5 days post
operatively.
Determination of mitotic index as a measure of
cytotoxicity
The mitotic index was used to determine if the
implantation of the biomaterial produced any
cytotoxicity. About 5 to 6 slides were prepared
from each blood culture and a total of 1000
lymphocytes were counted per culture to
determine the mitotic index. The mitotic index
was calculated as follows:
Number of lymphocytes in metaphase
Mitotic Index = -----------------------------------------------x 100
Total number of lymphocytes counted
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Archives of Orofacial Sciences 2006; 1: 15-20
Figure 1.
Implantation of synthetic hydroxyapatite granules (500 mg) in the right femur of a New
Zealand White rabbit (Oryctolagus cuniculus).
Figure 2.
Incubation of cultures at 37˚C for 72 hours in disposable centrifuge tubes containing rabbit
(Oryctolagus cuniculus) whole blood plus growth media.
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Kannan et al.
the chromosome spreads taken before
implantation (Fig 3) and after implantation (Fig
4) of the biomaterial are presented.
Determination of chromosomal aberrations
The slides were screened for both the numerical
and structural aberrations in all the blood samples
collected prior to and after the implantation of the
biomaterial. A minimum of 200 metaphase
chromosome spreads per sample was assessed to
score for any chromosomal aberrations that may
be induced due to the implantation of the
biomaterial.
DISCUSSION
The recent developments in the use of resorptive
biomaterials as osseous substitutes show a
growing interest by the potential users like
orthopaedic and maxillo-facial surgeons,
aestheticians, etc. (Braye et al,. 1996).
Hydroxyapatite has been widely applied to
biomaterials for bone tissue implantation, due to
its good biocompatibility and bioactivity as well
as the similarity to the inorganic component of
the hard tissues in natural bones (RodriguezLorenzo and Vallet-Regi, 2000). Calcium
hydroxyapatite microspheres suspended in an
aqueous gel carrier have been used mostly as
bone and dental substitution biomaterial. They act
as an osteoconductive scaffold for bone
reconstruction or as a bulking agent in soft tissues
(Glavas, 2005). If such materials are intended for
use in medicine, the combination of two different
mutagenesis studies (e.g. bacteria and
mammalian cells) continues to be necessary.
Many of them are based on the principle that
genotoxicity of mutagenicity serves as an
indicator for the carcinogenic potential of the
substance (Constantino et al., 1993).
The culture technique adopted in this
study using 0.3 ml of blood yielded consistent
results which are in accordance with the method
reported by the earlier workers (Stranzinger et al.,
1974; Chan et al., 1977; SchrÖder et al., 1978;
SchrÖder and Van Der Loo, 1979). Painter
(1926) was the first to report on the number of
chromosomes in rabbit as 44, which has then
been confirmed by many other authors (SchrÖder
et al., 1978; SchrÖder and Van Der Loo, 1979;
Williams and Ray, 1965). The modal
chromosome number of rabbit (Oryctolagus
cuniculus) in the present study was found to be
2n=44, XY.
Digital imaging
The slides were screened under oil objective
(100x) of Nikon Eclipse E200 light microscope
and the chromosomes were photographed using
Nikon Coolpix 995 digital imaging camera.
RESULTS
RPMI 1640 medium was found to be good for
culturing whole blood of rabbits for the
production
of
metaphase
chromosomes.
Colcemid added 1½ hours prior to harvesting the
cultures yielded good population of metaphase
chromosomes. The hypotonic treatment for 30
minutes using potassium chloride solution (0.56
per cent) was sufficient for swelling the cells.
Methanol and acetic acid used in the ratio of 3:1
for fixation yielded consistent results.
The modal chromosome number of rabbit
(Oryctolagus cuniculus) was found to be 2n=44,
XY. An optimum mitotic index of 3.30 ± 0.66
and 3.24 ± 0.27 per cent (Table 1) was obtained
before and after implantation of the biomaterial,
synthetic hydroxyapatite granules respectively. A
total of 1000 lymphocytes were counted per
culture to determine the mitotic index values.
A minimum of 200 chromosome spreads
at metaphase stage per sample was assessed to
score for any gross chromosomal abnormalities
that may be induced due to the implantation of
porous form of synthetic hydroxyapatite granules.
However, no gross chromosomal aberrations,
both numerical and structural were noticed in the
cultures set up, either prior to or after
implantation of the biomaterial. The pictures of
Table 1.
Mean Mitotic Indices of Lymphocytes Cultured from Five Rabbits.
Mean ± SE
Parameter
n
Before implantation of synthetic
hydroxyapatite granules
After implantation of synthetic
hydroxyapatite granules
Mitotic Index (%)
1000
3.30 ± 0.66
3.24 ± 0.27
n = number of lymphocytes / sample
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Archives of Orofacial Sciences 2006; 1: 15-20
Figure 3.
Metaphase chromosome spread of a male New Zealand White rabbit (Oryctolagus
cuniculus) before implantation of synthetic hydroxyapatite granules.
Figure 4.
Metaphase chromosome spread of a male New Zealand White rabbit (Oryctolagus
cuniculus) one week after implantation of synthetic hydroxyapatite granules.
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Kannan et al.
Chan FPH, Sergovich FR and Shaver EL (1977).
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(1998). In Vitro Chromosome Aberration Test –
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Rodriguez-Lorenzo LM and Vallet-Regi M (2000).
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Apatites. Chem Mater, 12: 2460–2465.
Sartorius DJ (1986). Coralline hydroxyapatite bone
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The mitotic index obtained before
implantation is 3.30 ± 0.66 per cent and after
implantation is 3.24 ± 0.27 per cent. The values
of mitotic index are an indication of the degree of
cytotoxicity. A reduction greater than 50 per cent
in the mitotic index value when compared to the
control indicates the cytotoxic nature of the test
substance (Health Effects Test Guidelines, 1998).
In the present study, there is no reduction in the
mitotic index value greater than 50 per cent
between the treatment (3.24 ± 0.27 per cent) and
control groups (3.30 ± 0.66 per cent).
No gross chromosome aberrations, both
numerical and structural were noticed either prior
to or after implantation. All the chromosome
spreads that were scored, both in the control and
treatment groups had a normal complement of
2n=44, XY without any structural aberrations.
This indicates that the result is negative and the
test substance, synthetic hydroxyapatite granules
(porous form) does not induce any gross
chromosome aberrations in cultured mammalian
cells. The mitotic index values and chromosomal
analyses in this study indicate that the test
substance, synthetic hydroxyapatite granules
(porous form) do not induce cytotoxicity nor
gross chromosomal abnormalities under the
present test conditions in New Zealand White
rabbits.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the USM
Short term grant 304/PPSG/6131299 for the
financial assistance, the staff of Tissue Bank and
also Miss Rodiah Mohd. Radzi and Mr. Shaharol
Anuar Abd. Latif for their help rendered in
animal house during this study.
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