Synthesis of Ca-hydroxyapatite Bioceramic from Egg Shell and its

Characterization
Samina Ahmed*
a
and Mainul Ahsan
a
Institute of Glass and Ceramic Research and Testing, Bangladesh Council of Scientific and
Industrial Research (BCSIR), Dr. Quadrat-i-Khuda Road, Dhanmondi, Dhaka - 1205,
Bangladesh
Abstract
Ca-hydroxyapatite (HA) was synthesised from egg shell under different conditions and characterised
by using AAS, FT-IR, XRD, SEM and EDS techniques. The results of these analyses combined togeth-
er indicate that sintered HA sample1 resembles the feature of pure and single apatite phase having
hexagonal structure. A Ca/P molar ratio of 1.69 was achieved in this case. On the other hand HA
obtained from calcined egg shell is a combination of both amorphous and crystalline phase and revealed
a sub-stoichiometric Ca-deficient apatite form with a Ca/P ratio of 1.50.
Keywords: Ca-hydroxyapatite, Biomaterials, Hexagonal, stoichiometric apatite, Ca-deficient apatite
Bangladesh J. Sci. Ind. Res. 43(4), 501-512, 2008
Introduction
Recently, applications of bioceramic materi-
als in biomedical fields has received consid-
erable attention (Feng et al. 2005, Xu et al.
2001, Balzsi et al. 2007, Sasikumar et al.
2006) and researchers are now actively
engaged in developing many routes to syn-
thesis Ca-phosphate based bioceramic mate-
rials using different sources of calcium (Ca)
and phosphate (P) (Feng et al.2005, Xu et al.
2001, Balzsi et al. 2007, Sasikumar et al.
2006). Bioceramics are required to alleviate
pain and restore function of diseased and
damaged body parts. Ca-phosphate based
synthetic ceramic materials, particularly, Ca-
hydroxyapatite (HA), -tricalcium phos-
phate (-TCP) and their composites are
extensively used for medical purposes that
includes tissue engineering, implants, coat-
ing on prostheses and bone filling materials
(Prabakaran et al. 2005). However, among
these bioceramics, HA, Ca
10
(PO
4
)
6
(OH)
4
, is
an important inorganic material in both biol-
ogy and chemistry (Markovic et al. 2004)
and due to its excellent bioactive, biocom-
patible nature and bone bonding ability with
surrounding tissues it is widely used in tissue
File 2792, 43(4) 2nd Print (7)
* Correspondence author, E-mail: [email protected],[email protected]
BCSIR
Avaiable online at www.banglajol.info
BANGLADESH JOURNAL
OF SCIENTIFIC AND
INDUSTRIAL RESEARCH
E-mail: bjsir07gmail.com
502 Synthesis of Ca-hydroxyapatite Bioceramic 43(4) 2008
engineering and bone replacement (Balzsi
et al. 2007). It is well known that human
bone is a hybrid composition of inorganic
(~70% apatitic calcium phosphate with a
Ca/P ratio 1.66) and organic (~30% colla-
gen) materials. The demands for synthetic
HA as a substitute material for bone and
teeth in orthopedic and dentistry fields are
increasing day by day. Considering the
numerous applications of HA in biomedical
fields, development of various synthesis
methods is a major issue now and obviously,
a simple cost-effective procedure is highly
desirable to the researchers. A number of
synthesis techniques using various sources
of Ca and P have been developed which
includes wet chemical method (precipita-
tion), sol-gel method, hydrothermal synthe-
sis procedure, continuous precipitation, ther-
mal deposition and solid state reaction
method (Feng et al. 2005, Balzsi et al.
2007, Thamaraiselvi et al. 2006). However,
among these methods, wet chemical precipi-
tation route is popular because of its simple,
cheap and easy application in industrial pro-
duction.
Although the demand of wet precipitation
method scores high, the choice of raw mate-
rials to use as Ca and Pprecursors is the key
factor in any method to be developed. From
this point of view, in this work we have
adapted wet chemical method to synthesize
pure HA bioceramics with Ca/P ratio 1.66,
using egg shell as Ca source. Depending on
feeding egg shell contains about (94 - 97) %
CaCO
3
while rest of the percentage is organ-
ic matter and egg pigment. Egg shell is a
waste material after the usage of egg. Hence,
utilisation of egg shell will benefit in two
ways: (i) egg shell derivated HAwill be cost-
effective bioceramic material for biomedical
applications, and (ii) utilisation of egg shell
will be an effective material-recycling tech-
nology for waste management.
Materials and Methods
Synthesis of HA
At first the raw egg shells were cleaned thor-
oughly with water followed by boiling for
hr. It was then dried overnight at 110
O
C in an
oven and then crushed and powdered using
an agate mortar. This egg shell powder was
then divided into two portions: one portion
remained uncalcined and percentage of
CaCO
3
(~94%) was determined via titration
method, while the 2nd portion was calcined
at 950
O
C to burn out the organic and other
substances. Both the uncalcined and calcined
egg shell powder was used to synthesize HA
according to the following procedure.
Astoichiometric amount of egg shell powder
(both uncalcined and calcined) was dis-
solved in conc. HNO
3
and15 mL of distilled
water was added to the acidic egg shell solu-
tion and filtered to get clear solution. Final
volume was made up to 50 mL with distilled
water and the pH of the solution was
increased to ~10.0 with aqueous ammonia.
A requisite amount of 0.10 M Na
2
HPO
4
.
12H
2
O in ammonia solution (pH ~10.0) was
Ahmed and Ahsan 503
added drop wise to the egg shell solution
with continuous stirring condition. White
gelatinous precipitate of HA was formed
which was stirred for overnight in the moth-
er solution for ripening. The precipitate was
filtered through a Buchner funnel and thor-
oughly washed with plenty of distilled water.
At this stage, the filtered precipitate was
dried at 110
O
C to remove any trace of water.
The HA (sample 1) synthesised from uncal-
cined egg shell was then subjected to calci-
nation at 900
O
C for hr, while the HA(sam-
ple 2) obtained from calcined egg shell
remained as received, i.e. no further calcina-
tion was done, but crushed to achieve fine
powder. A flow diagram of the synthetic pro-
cedure is given in Figure 1.
All the chemicals used for this study were
Analar grade either from E. Merck or BDH
Fig. 1. Flow diagram of hydroxyapatite synthesis procedure from egg shell
504 Synthesis of Ca-hydroxyapatite Bioceramic 43(4) 2008
and the solutions were prepared using dou-
ble distilled water.
Characterisation of synthesised HA powders
The semi quantitative elemental information
of the samples was achieved by energy dis-
persive spectroscopy (EDS) as a preliminary
technique. Then chemical analysis was done
to determine the percentage of Ca and P by
atomic absorption (AAS) and UV spec-
trophotometric methods respectively.
Fourier transform infrared spectroscopy (FT-
IR, Model no. FT-IR - 8900, SHIMADZU)
was used to identify the functional groups.
Experimental spectra were obtained by using
KBr disks with a 1:100 "samples-to-KBr"
ratio and the samples were scanned in the
wave number range of 4000 cm
-1
- 400 cm
-1
with an average of 30 scans. The resolution
of the spectrometer was 4 cm
-1
. Phase purity
of the synthesised samples was examined by
using PANalytical (X'Pert PRO XRD PW
3040). The intensity data were collected in
0.02
O
steps following the scanning range of
2 =20
O
- 80
O
using CuKa ( =1.54178
O
A)
radiation. The phases developed in both the
samples were compared and confirmed
using standard J CPDS files (card no. 89-
6439). The surface morphology and micro-
structural features of the samples were
observed by Hitachi, S-3400N scanning
electron microscopy (SEM) equipped with
energy dispersive spectroscopy (EDS).
Results and Discussion
The EDS spectra of HA samples are shown
in Figures 2a and 2b respectively. The EDS
spectrum of HA(sample 2) clearly shows the
peaks of Ca, P, C and O as expected. The
presence of C is quite usual in this case as
the sample is only oven dried at 110
O
C,
whereas in case of HA sample 1 which was
calcined at 900
O
C, there is no trace of C.
This observation was further validated by
FT-IR and XRD results. The Ca/P molar
ratio (1.74 and 1.499 for sample 1 and 2
respectively) was qualitatively calculated
using EDS data. However, the chemical
assessment provided quantitative informa-
tion regarding the Ca/P molar ratio of the
HA samples. A ratio of 1.69 confirmed the
stoichiometric nature of HAsample 1 where-
as Ca/P molar ratio of 1.50 showed the sam-
ple 2 as sub-stoichiometric calcium deficient
HA.
The FT-IR spectra of the HA samples are
shown in Figures 3a and 3b. The band posi-
tions and their respective assignments are
given in Table I. The IR spectra of both the
HA samples (1 and 2) exhibited well-defined
absorption bands in the range of 962 ~1040
cm
-1
and 565 ~635 cm
-1
, characteristic of a
well- crystallized apatite phase (Xu et al.
2001). The peaks appeared at 1039.6 cm
-1
(HA sample 1) and 1035.7 cm
-1
(HA sample
2) are assigned to asymmetric stretching
vibration (v
3
) of PO
4
3-
groups, while the
peaks at 962.4 cm
-1
corresponds to the sym-
Ahmed and Ahsan 505
Fig. 2. EDS spectra of synthesised HA (a) HA sample 1 (b) HA sample 2.
506 Synthesis of Ca-hydroxyapatite Bioceramic 43(4) 2008
Fig. 3. FT-IR spectra of synthesised HA (a) HA sample 1 (b) HA sample 2.
Ahmed and Ahsan 507
metric stretching (v
1
) mode of PO
4
3-
.
Bending (v
3
) mode of PO
4
3-
group is
observed at positions 474.5 cm
-1
and 472.5
cm
-1
for samples 1 and 2 respectively (Tas
2000). The sharp and peaks at 601.7 and
570.9 cm
-1
for sample 1 and 603.7 and 565.1
cm
-1
for sample 2 are assigned to the bending
mode of PO
4
3-
. The noticeable large separa-
tion of these two bands further suggests the
presence of crytallized apatitic phase
(LeGeros 1991, Tas 2000). The IR spectra of
the HA sample 2 (Fig. 3b) resembles the
nature of hydrated carbonated apatite. The
absorption bands observed in the region
1419.5 cm
-1
and 873.7 cm
-1
are due to the
vibrational mode of CO
3
2-
group associated
in amorphous phase (Xu et al. 2001). This
observation is consistent with the XRD
results. However, in case of calcined HA
(Sample 1, calcined at 900
O
C), there is no
noticeable bands for CO
3
2-
group (Fig. 3a)
indicating the elimination of CO
3
2-
due to
the release of volatile gas. This sample also
shows a sharp band at 632.6 cm
-1
, which is
indicative of the presence of structural OH-
(OH liberation mode), whereas in the IR
spectrum of HA sample 2, the same band is
not visible. This result attributed to the for-
mation of good crystallinity for the HA sam-
ple 1 after calcination at 900
O
C which is also
evidenced from the corresponding XRD data
(Fig. 4a). The bands observed at 1635.5 cm
-
1
and 3361.7 cm
-1
in HA sample 2 confirms
the presence of adsorbed H
2
O which is quite
usual since this sample remains in uncal-
cined state. On the other hand in case of sam-
ple 1, these bands are very weak indicating
the loss of H
2
O molecules during calcina-
tion, but the additional band at 3500 cm
-1
clearly represents the hydroxyl stretching
mode associated with surface P-OH groups
(Mahabole et al. 2005).
474.5
570.9
601.7
632.6
----
962.4
1039.6
---
1650.0
3500.0
472.5
565.1
603.7
----
873.7
962.4
1035.7
1419.5
1635.5
3361.7
PO
4
3-
bending (v
2
)
PO
4
3-
bending (v
4
)
PO
4
3-
bending (v
4
)
OH liberation (Structural OH-)
CO
3
2-
group
PO
4
3-
stretching (v
1
)
PO
4
3-
bending (v
3
)
CO
3
2-
group (v
3
)
H
2
O adsorbed (v
2
)
Structural OH-
Table I : FT-IR band positions and their corresponding assignments
Observed band positions (cm
-1
)
HA sample 1 HA sample 2
Corresponding assignments
508 Synthesis of Ca-hydroxyapatite Bioceramic 43(4) 2008
Atypical XRD diffraction of sample1 is pre-
sented in Figure 4a. The major d spacing val-
ues are compared with the J CPDS standard
data (Ref. code: 89-6439) for HAP and are
summarized in Table II. A good agreement
between the experimental data (achieved for
sample 1) and J CPDS standard for calcium
hydroxyapatite (Ref. code: 89-6439) both in
terms of intensity and d-spacings (Table II)
was obtained. Particularly the strong diffrac-
tion peaks corresponding for hydroxyapatite
at 2 position 31.7858 (211) plane together
with other two peaks at 32.2665
O
and
32.9515
O
of almost equal intensities con-
firmed the formation of pure hydroxyapatite
in crystallite form [19]. However no peak
was detected at 29.399
O
for calcium carbon-
ate with HA phase (Vijayalakshmi et al.
2006) which also satisfies the IR spectral
data for HA sample 1 where the elimination
of CO
3
2-
peak has already been observed.
Figure 4. XRD spectra of synthesised HA (a) HA sample 1 (b) HA sample 2.
Ahmed and Ahsan 509
The crystallite sizes for HA sample 1 were
calculated from Scherrer's relationship D =
79.5/ cos , where D =crystal size (
O
A),
=FWHM in degree.
The Bragg reflection at (211), (112) and
(300) planes of this sample were considered
to calculate the crystallite size. The sizes of
the crystallites for these phases are 41 nm,
65 nm and 34 nm respectively. The lattice
parameters of this HA sample 1 were meas-
ured as a =b =9.4164
O
A and c =6.8789
O
A
which are very close to those values obtained
from the J CPDS (ref. code: 89-6439) stan-
dard data (a =b =9.4172 A and c =6.8799
A) and the lattice parameters of human
bones (a =b =9.419
O
A and c =6.88
O
A)
d-spacings (
O
A) Intensity (%) (h k l)
Experimental J CPDS Experimental J CPDS
4.0748
3.8999
3.4395
3.1673
3.0861
2.8152
2.7744
2.7183
2.6296
2.5271
2.2619
2.1482
2.0610
1.9443
1.8914
1.8413
1.8060
1.7806
1.7538
1.7212
1.6447
1.4742
4.0777
3.8885
3.4399
3.1695
3.0825
2.8131
2.7776
2.7185
2.6293
2.5282
2.2619
2.1487
2.0617
1.9428
1.8899
1.8399
1.8054
1.7797
1.7539
1.7199
1.6436
1.4738
6.38
4.45
38.68
8.36
14.60
100.00
59.40
55.42
23.44
5.30
19.31
5.28
5.54
28.19
14.58
32.19
15.67
11.83
12.30
15.73
4.41
6.99
6.40
5.80
35.50
8.50
14.15
100.00
47.60
64.20
21.90
4.00
22.20
5.50
5.20
27.60
11.70
30.30
16.00
11.00
11.90
13.70
5.60
7.40
2 0 0
1 1 1
0 0 2
1 0 2
2 1 0
2 1 1
1 1 2
3 0 0
2 0 2
3 0 1
1 3 0
1 3 1
1 1 3
2 2 2
1 3 2
2 1 3
3 2 1
1 4 0
4 0 2
0 0 4
3 2 2
5 0 2
Table II. Indexed d-spacings (in hexagonal unit cell) of XRD data obtained for synthesised HA
sample 1
510 Synthesis of Ca-hydroxyapatite Bioceramic 43(4) 2008
(LeGeros et al. 1993). This good agreement
between the experimental data and standard
values reflects that the synthesized HAP
sample resembles the feature of pure and sin-
gle apatite phase having hexagonal structure.
The formation of apatite of single phase also
confirmed the Ca/Pmolar ratio 1.66 which is
very close to that of crystalline apatite (Elliot
et al. 1994, Xu et al. 2001).
Since sintering temperature or calcination
effect plays an important role in the forma-
tion of crystallite phase (Feng et al. 2005),
the XRD profile of the as received HA
sample (i.e. after oven dried at 110
O
C) was
recorded and displayed in Figure 4b.
Although the formation of hydroxyapatite
was confirmed by comparing the experimen-
tal data with the J CPDS standard file (ref.
code: 89-6439), but it is cleared from the
XRD pattern that the HA sample under
investigation has formed in poorly crys-
talline phase together with amorphous
phase supporting the IR informations. This
behaviour can be attributed to the tempera-
ture effect, since this sample 2 remained in
only oven dried state. It is well established
that the increasing of sintering temperature
Figure 5. SEM micrographs of synthesised HA sample 1 (a and b) magnification 2000
(c) magnification 180 and (d) magnification 350.
Ahmed and Ahsan 511
increases the crystallite degree resulting sev-
eral distinct peaks for HA (Feng et al. 2005).
So the low crystallinity and amorphous
nature can be upgraded or improved to a
solid, crystalline phase via the thermal treat-
ment (Santos et al. 2004).
The morphology and micro structural fea-
tures of the HA samples (both 1 and 2)
recorded at different magnifications are
shown in SEM micrographs. The micro-
graphs (Fig. 5a - 5d) representing the
micro structural features of HA sample 1,
exhibit a combination of spherical, hexago-
nal and cylindrical shapes having crystal size
of ~30 - 60 nm. On the other hand, the dif-
ferent magnified micrographs of HA sample
2 (Fig. 6a - 6b) does not shows clear crystal-
lite phases since the sample was a combina-
tion of amorphous and crystallite phase.
Conclusions
Ca-hydroxyapatite nano powders have been
successfully synthesised using calcined and
uncalcined egg shell as the prime source of
Ca. The results showed that HA sample 1
consists of single crystallite apatite phase
and sample 2 contains both amorphous and
crystalline phase. The crystallinity of this
later sample can be improved by further sin-
tering of the product. However, the egg shell
derivated HA will no doubt be a cost-effec-
tive bioceramic material for biomedical
applications. Additionally, utilisation of egg
shell will be an effective material-recycling
technology for waste management.
Acknowledgement
We thank Dr. Dilip Kumar Saha of Atomic
Energy Commission for XRD, Mr.
Fariduddin Farhad and Mr. Shudhangsu
Kumar of BCSIR for SEM and FT-IR
respectively. Financial support from IGCRT,
BCSIR is gratefully acknowledged.
Fig.6. SEM micrographs of synthesised HA
sample 2 (a) magnification 500 (b)
magnification 2000.
512 Synthesis of Ca-hydroxyapatite Bioceramic 43(4) 2008
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Received : July 02, 2008;
Accepted : August 21, 2008