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Synthesis and Characterization of Calcium
Phosphate/Chitosan Composites
Kiagus Dahlan1, Setia Utami Dewi1, Ai Nurlaila2, Djarwani Soejoko3
1
Department of Physics, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Indonesia
2
Department of Science Education, Faculty of Education, State Islamic University, Jakarta, Indonesia
3
Department of Physics, Faculty of Mathematics and Natural Sciences, University of Indonesia, Indonesia
Abstract
Synthesis of biomaterial composites consisting of organic matrix and inorganic mineral has been
performed. The inorganic minerals used were hydroxyapatite and hydroxyapatite/carbonate
apatite compounds. These apatites were resulted from calcium of chicken eggshell calcinated at
1000oC and phosphor of diammonium hydrogen phosphates. The organic matrices used were
chitosan originated from shrimp shells. To produce the composites, both apatites were mixed
with chitosan. The resulted composite samples were further dried at 50°C. Two types of
composition used in this experiment were 20% chitosan mixed with 80% calcium phosphate and
30% chitosan mixed with 70% calcium phosphate. In each composition, the calcium phosphate
ceramics consisted of hydroxyapatite and hydroxyapatite/carbonated apatite. The X-ray
diffraction profiles showed that chitosan did not change the calcium phosphate phases. Chitosan
seemed only influence the crystallinity of the compound in which an increase of chitosan
decreased the crystallinity. The scanning electron microscope (SEM) micrographs clearly
showed that the surface morphology of hydroxyapatite/carbonated-chitosan composites with the
composition of 64% HA/16% HA/CA/20% chitosan were in the form of granules. Samples
containing more carbonate apatites showed more granules.
Keywords: biomaterial, hydroxyapatite , apatite, diffraction, crystallinity
Introduction
Natural bone consists of organic and inorganic substance. Organic substance possesses important
property for the growing of bone minerals, whilst inorganic substance supports the mechanical
strength of the bone. Calcium phosphate is a major component in bone minerals [1-5].
Hydroxyapatite (HA), the most commonly used calcium phosphate in medical fields, possesses
excellent biocompatibility and is osteoconductive [6,7]. It has the chemical formula
Ca10(PO4)6(OH)2 and a Ca/P molar ratio of 1.67. Presence of carbonate (CO3) may substitute
either for the hydroxyl (OH) or the phosphate (PO4) groups, designated as type A or type B
carbonate apatites, respectively. The substitutions cause morphological changes in precipitated
apatite crystals as well as their properties. Carbonate substituted apatite is more soluble than
carbonate-free synthetic apatite [8-10]. High brittleness and low tensile strength of these
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bioactive materials can be reduced by the formation of the carbonate apatite (CA) on the surface
of biomaterials.
HA can be synthesized using various methods [11,12]. Calcium nitrate tetrahydrate
[Ca(NO3)24H2O] and diammonium hydrogen orthophosphate [(NH3)2HPO4] had been used to
form HA using precipitation method [13]. It was found that the crystallinity increased with the
temperature of calcination. At calcination temperature of 1000oC, as shown by X-ray diffraction
(XRD) patterns, the presence of the peaks of dicalcium phosphate and tricalcium phosphate was
observed. Hydrothermal synthesis with a temperature of 200oC for 24 hours showed pure
hydroxyapatite, but after 48 hours dicalcium phosphate was also detected [14].
HA has also been synthesized from synthetic and some natural calcium sources. Common
calcium synthetic sources are Ca(OH)2, Ca(NO3)24H2O and CaCl22H2O [15,16]. Several natural
calcium sources that have been used are coral, marine algae, bovine, and seashells [17-22]. In
vitro and in vivo studied of natural apatite from compact bone and synthetic HA showed that
natural apatite was well tolerated and had good osteoconductive [23]. Therefore, it is reasonable
to assume that the use of natural sources of calcium may increase biocompatibility of the
biomaterials.
In this resarch, apatite/chitosan composites were synthesized from chicken eggshells as the
calcium precursor and chitosan as the inorganic matrix. Chitosan is natural biopolimer obtained
by chemical deacetilation of chitin from shrimp or crabs which has reactivity as chemical bond
and the structure resembles organic matrix bones. Chitosan is also biodegradable, biocompatible,
non antigenic, and osteoconductive. Synthesis of calcium phosphate/chitosan might be
performed by several methods like precipitation, sol-gel and cement formation [24-28]).
Composite synthesized in this study consisted of HA, HA/CA mixture and chitosan to get the
soluble and mechanically strong biomaterials.
Experimental Procedure
Two steps of experiments were performed in this research. The first step was to synthesize HA
as a stable crystal of calcium phosphate and CA as the more soluble crystal. This step aimed to
get HA and HA/CA mixture. The second step was to synthesize and characterize calcium
phosphate/chitosan composite.
Starting materials used for HA synthesis in this experiment were chicken eggshells and
diammonium hydrogen phosphates [(NH4)2HPO4]. Eggshells were cleaned and heated at 1000°C
for 5 hours to remove their organic components and decompose the calcium carbonate into
calcium oxide. Calcium phosphate compound was synthesized using precipitation method by
adding 100 ml [(NH4)2HPO4] solution into 100 ml CaO suspension. The solution was maintained
at a temperature of 37oC and stirred with a magnetic stirrer during the addition processes. The
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resulting precipitate was then allowed to settle for 24 hours at room temperature and some water
was permitted to evaporate. The powder obtained was dried in an oven at 110oC for 5 hours.
Concentrations of Ca precursor, between 0.1 M and 0.5 M, and P precursor, between 0.06 M and
0.3 M, were varied stoichiometrily keeping the ratio of Ca/P being 1.67.
Calcium phosphate-chitosan composite was synthesized using sonication stirrer. Calcium
phosphate used were HA and HA/CA mixture. 20% chitosan and 80% HA and HA/CA mixture
were performed using sonication method by stirring the solution with ultrasonic wave 42 kHz
130 W for 3 hours. After sonication processes, the solution was allowed to settle for 12 hours at
room temperature. The sampel obtained was then dried at 50oC for 15 hours.
Two methods of characterization were applied to the samples, X-ray diffraction (XRD) and
scanning electron microscopy (SEM). XRD characterization used PHILIPS model APD 3520
diffractometer. It had Cu target, so the resulted X-ray had wavelength 1.54060 x 10-10 m. SEM
characterization used JEOL JCM-35C scanning electron microscope. Samples were coated first
by gold-palladium (80% Au and 20% Pd). Coating processes used Ion Sputter JFC-1100
machine.
Results and Discussion
Synthesis of calcium phosphate compounds was started from calcinating chicken eggshells at
temperature 1000oC for 5 hours. This procedure resulted in CaO and elimination of some organic
components of the samples. Approximately 34% sample mass was eliminated. The procedure
was continued by precipitating CaO solution into (NH4)2HPO4 solution to produce calcium
phosphate compounds. The resulted powder had mass percentage of about 62-65%. Figure 1
shows X-ray diffraction patterns of HA synthesized from various concentration of CaO solution
derived from calcinated eggshells and (NH4)2HPO4 solution. This identification referred to
JCPDS 09-0432 data for HA. Pure HA X-ray diffraction patterns was resulted from combination
of 0.3 M Ca and 0.18 M P precursor. Combination of 0.5 M Ca and 0.3 M P precursor showed
the presence of HA and TCP. Therefore, it might be concluded that the optimum concentrations
to obtain pure HA were 0.3 M Ca and 0.18 P precursor. Higher concentrations showed the
presence of other calcium phosphate phases. This pure HA patterns resulted in the HA crystal
size of about 33 nm which is similar with the size of high crystalline HA crystal [29]. HA crystal
has hexagonal rhombic prisms with the lattice parameters being a = b = 0.9418 nm and c =
0.6881 nm. By using Cohen method, the lattice parameters of HA samples obtained in this
experiment were found to be a = b = 0.9420 nm and c = 0.6881 nm which were very close to
JCPDS data.
Addition of CA was observed to lower the intensity and wider the peaks of diffraction patterns as
shown in Figure 2. This wider but same position of peaks indicated that the sample consisted of
HA and CA. The widening of the X-ray diffraction pattern peaks may indicate that the crystal
size was smaller which also meant that the solubility of the sample should be higher. Therefore,
combination of stable and brittle crystal HA and soluble HA/CA mixture was thought to be
excellent choice to make mineralization processes in bone run very quick. Pure HA might be
excellent bone filler in small bone damage, but for serious fracture, pure HA may not be enough.
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1400
400
311
202
301
600
210
102
800
212
1000
(a)
112
300
002
1200
113
1600
Intensity (counts/s)
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0
1600 25
30
35
40
45
1400
(b)
1200
1000
800
600
400
x
200
0
25
30
35
40
45
o
2θ ( )
Figure 1 X-ray diffraction patterns of HA synthesized by using (a) 0.3 M Ca and 0.18 M P and (b) 0.5 M
Ca and 0.3 M P. Higher concentration of (NH4)2HPO4 resulted in the presence of β-TCP
It must be modified to enhance its mechanical strength characteristics. In this investigation, HA
was combined with HA/CA mixture and chitosan to reduce brittle characteristics of HA and
make strong, more soluble and more biodegradable biomaterial composites.
Synthesis of calcium phosphate/chitosan composites were done by dispersing HA and CA
particles into chitosan fibers. Effects of chitosan on the crystallinity of HA and HA/CA samples
were examined by adding 20% and 30% chitosan in samples. It was found that the addition of
chitosan in HA samples and HA/CA samples showed similar results. This addition did not
change the position and the number of peaks in X-ray diffraction patterns. The only effect
observed was a slightly change in the intensity of the peaks. Higher concentration of chitosan in
samples seemed correspond to the lower intensity of the peaks. The peaks were also observed
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350
Intensity (counts/s)
300
(a)
250
200
150
100
50
0
1600 25
30
35
40
45
1400
1200
(b)
1000
800
600
400
200
0
25
30
35
40
45
2θ (o )
Figure 2 X-ray diffraction patterns of (a) CA and (b) HA
wider. Figure 3 showed X-ray diffraction patterns of HA/chitosan composite with different
percentage of chitosan, 20% and 30%. It was observed that higher percentage of chitosan
resulted in the lower intensity in the X-ray diffraction patterns of the composites sample. 30%
chitosan in the sample resulted in the lowering intensity of one fourth compared to the chitosan
percentage of 20%. The amorphous characteristics of chitosan also reduced the crystallinity of
HA. However, it could be seen as`well that different percentages of chitosan did not change any
phases in the patterns.
Optimum composition of HA, HA/CA and chitosan has not been found yet. Here, two types of
combination had been investigated, 20% chitosan combined with 80% calcium phosphate
compound and 30% chitosan combined with 70% calcium phosphate compound. Calcium
phosphate ceramics consisted of HA and HA/CA mixture was also varied, 80% HA combined
with 20% HA/CA mixture and 70% HA combined with 30% HA/CA mixture. This investigation
presented that 20% chitosan in the composite was optimum. It was observed that if the
percentage was higher, some of the chitosan did not make any interaction with the apatite and,
therefore, it seemed to dominate the composite. 20% chitosan was the most effective percentage.
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(a)
8 00
6 00
4 00
2 00
0
15
20
25
30
35
40
45
80 0
(b )
60 0
40 0
20 0
0
15
20
25
30
35
40
45
o
2θ ( )
Figure 3 X-ray diffraction patterns of HA/chitosan composite with percentage of HA:chitosan of (a)
(80:20)% and (b) (70:30)%
It was also observed here that 20% HA/CA mixture combined with 80% HA seemed to be the
best composition of calcium phosphate ceramics as the more percentage of HA/CA mixture
might make the crystal size too small and the sample may vanish very quick in body solution.
HA crystal size was obtained to reduce from 33.33 nm to 18.32 nm for plain (002) and 33.84 nm
(a)
(b)
(c)
Figure 4 SEM micrographs of calcium phosphate-chitosan composites with the percentage of HA:HACA:chitosan of (a) (64:16:20)%, (b) (56:24:20)% and (c) (0:80:20)%
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to 20.91 nm for plain (300) by addition of 20%-30% chitosan in samples. For HA/CA samples,
addition of chitosan apparently reduced the crystal size from 15.01 nm to 12.84 nm for plain
(002) and 15.11 nm to 9.26 nm for plain (300).
Surface morphology of chitosan showed small pores of about 0.1 to 0.2 µm in diameter, whilst
surface morphology of HA showed crystals in the forms of bars of about 0.3 µm long. Surface
morphology of HA/CA mixture showed collection of granules with the size of about 02 µm. It
was not found that addition of 20% chitosan change the surface morphology of samples. SEM
micrographs of calcium phosphate/chitosan composites with the percentage of HA, HA/CA
mixture and chitosan varied were presented in Figure 4. The surface morphology of 64%
HA/16% HA/CA mixture/20% chitosan composite showed granules with the size of about 0.2
µm long. It was also seen that 16% HA/CA and 24% HA/CA still presented separate particles of
granules, but the composites with 80% HA/CA did not show any separate particles.
Conclusions
Composite consisted of HA, HA/CA mixture and chitosan synthesized by using chicken
eggshells as starting materials could be developed as bone substitute biomaterial. This calcium
phosphate/chitosan composite was synthesized using sonication method by which the samples
were stirred in ultrasonic chamber with the frequency of 42 kHz and power of 130 W for 3
hours. This made the mixture more homogeneous.
Variation in percentage of chitosan in the composite did not change HA or HA/CA phases in the
composite, but it changed the crystal sizes. 20% of chitosan in the composite was observed to be
the most effective contribution of chitosan in the composite as some chitosan did not make any
interaction with the calcium phosphate if its percentage was higher. The optimum composition of
the composite obtained in this research was 64% hidroxyapatite, 16% HA-CA mixture and 20%
chitosan.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
C.T. Laurencin, A.M.A. Ambrosio, M.D. Borden, J.A. Cooper, Ann. Rev. Biomed. Eng.
1 (1999) 19.
R.D. Pilliar, M.J. Filiaggi, J.D. Wells, M.D. Grynpas, R.A. Kandel, Biomate. 22 (2001)
963.
T.K. Daftari, T.R. Whitesides, J.G. Heller, Spine. 19 (1994) 904.
J.L. Meyer, E.D. Eanes, Calcif. Tiss. Ress. 25 (1978) 59.
S. Takagi, M. Mathew, J. Res. Natl. Stand. Technol. 106 (2001) 1030.
S. N. Danilchenko, A.V. Koropov, I. Yu. Protsenko, B. Sulkio-Cleff, L. F. Sukhodub,
Cryst. Res. Technol. 41 (2006) 268.
N. Pleshko, A. Boskey, R. Mendelsohn, Biophys. J. 60 (1991) 786.
S.N. Danilchenko, V.A. Pokrovskiy, V.M. Bogatyrov, L.F. Sukhodub, B. Sulkio-Cleff,
Cryst. Res. Technol. 40 (2005) 692.
L. Muller, F.A. Muller, A. Bioamate. 2 (2006) 181.
123701-4848 IJBAS-IJENS © February 2012 IJENS
IJENS
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 01
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
57
R.Z. LeGeros, J.P. LeGeros, O.R. Trautz, W.P. Shirra, Adv. X-Ray anal. (1971) 57.
Jae-Kil Hana, Ho-Yeon Song, Fumio Saito, Byong-Taek Lee, Mat. Chem. Phys. 99
(2006) 235.
F. Chen, Z.C. Wang, C.J. Lin, Mater. Lett. 57 (2002) 858.
K. Deepak, D.P. Pattanayak, S. Upadhyay, R.C. Prasad, B.T. Rao, T.R. Rama Mohan,
Tren. Biomater. Artif. Organs 18 (2005) 87.
J.S. Earl, D.J. Wood, S.J. Milne, J. Phys. 26 (2006) 268.
E.C.Victoria, F.D. Gnanam, Trends Biomater. Artif. Organs. 16 (2002) 12.
L. Kong, Y. Gao, G. Lu, Y. Gong, N. Zhao, X. Zhang, Europ. Polymer J. 42 (2006) 3171.
M. Sivakumar, T.S.S. Kumar, K.L. Shantha, K.P. Rao, Biomate. 17 (1996) 1709.
G. Felicio, C.M. Laranjeira, Qui. Nov. 23 (2000) 441.
C.Y. Ooi, M. Hamdi, S. Ramesh, Ceram. Interna. 33 (2007) 1171.
K. Prabakaran, A. Balamurugan, S. Rajeswari, Bull. Mater. Sci. 28 (2005) 115.
S. Kenneth, Vecchio, X. Zhang, J. B. Massie, M. Wang, W.K. Choll, A. Biomate. 3
(2007) 910.
K.A. Vecchio, X. Zhang, J.B. Massie, M. Wang, C.W. Kim, A. Biomate. 23 (2007) 910.
G. Saraswathy, S. Pal, S. Rose, T.P. Sastry, Bull. Mater. Sci. 24 (2001) 415.
L. Kong, Europ. Polim. J. 42 (2006) 3171.
H. Liu, A. Biomate. 2 (2006) 557.
H.H. Jin, C.H. Lee, W.K. Lee, J.K Lee, Mater. Lat. 62 (2008) 1630.
O.C. Wilson, J.R. Hull, Mater. Sci. Eng. 28 (2008) 434.
I. Yamaguchi, S. Iizuka, A. Osaka, H. Monma, J. Tanaka, Coll. Surf. A: Physicochem.
Eng. Aspects. 214 (2003) 111.
N. Pishko, Boskey, Mendisohn, Biophys. J. 60 (1991) 786.
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