Synthesis and Characterization of Calcium Phosphate/Chitosan Composites
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1 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 1 5 Synthesis and Characterization of Calcium Phosphate/Chitosan Composites Kiagus Dahlan 1, Setia Utami Dewi 1, Ai Nurlaila 2, Djarwani Soejoko 3 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 1 o C 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 5 C. Two types of composition used in this experiment were 2% chitosan mixed with 8% calcium phosphate and 3% chitosan mixed with 7% 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/2% 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 Ca 1 (PO 4 ) 6 (OH) 2 and a Ca/P molar ratio of Presence of carbonate (CO 3 ) may substitute either for the hydroxyl (OH) or the phosphate (PO 4 ) 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 [8-1]. High brittleness and low tensile strength of these
2 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 1 51 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(NO 3 ) 2 4H 2 O] and diammonium hydrogen orthophosphate [(NH 3 ) 2 HPO 4 ] 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 1 o C, 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 2 o C 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(NO 3 ) 2 4H 2 O and CaCl 2 2H 2 O [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 [(NH 4 ) 2 HPO 4 ]. Eggshells were cleaned and heated at 1 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 1 ml [(NH 4 ) 2 HPO 4 ] solution into 1 ml CaO suspension. The solution was maintained at a temperature of 37 o C and stirred with a magnetic stirrer during the addition processes. The
3 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 1 52 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 11 o C for 5 hours. Concentrations of Ca precursor, between.1 M and.5 M, and P precursor, between.6 M and.3 M, were varied stoichiometrily keeping the ratio of Ca/P being Calcium phosphate-chitosan composite was synthesized using sonication stirrer. Calcium phosphate used were HA and HA/CA mixture. 2% chitosan and 8% HA and HA/CA mixture were performed using sonication method by stirring the solution with ultrasonic wave 42 khz 13 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 5 o C 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 352 diffractometer. It had Cu target, so the resulted X-ray had wavelength x 1-1 m. SEM characterization used JEOL JCM-35C scanning electron microscope. Samples were coated first by gold-palladium (8% Au and 2% Pd). Coating processes used Ion Sputter JFC-11 machine. Results and Discussion Synthesis of calcium phosphate compounds was started from calcinating chicken eggshells at temperature 1 o C 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 (NH 4 ) 2 HPO 4 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 (NH 4 ) 2 HPO 4 solution. This identification referred to JCPDS data for HA. Pure HA X-ray diffraction patterns was resulted from combination of.3 M Ca and.18 M P precursor. Combination of.5 M Ca and.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.3 M Ca and.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 =.9418 nm and c =.6881 nm. By using Cohen method, the lattice parameters of HA samples obtained in this experiment were found to be a = b =.942 nm and c =.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.
4 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: Intensity (counts/s) (a) (b) x θ ( o ) Figure 1 X-ray diffraction patterns of HA synthesized by using (a).3 M Ca and.18 M P and (b).5 M Ca and.3 M P. Higher concentration of (NH 4 ) 2 HPO 4 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 2% and 3% 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
5 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: Intensity (counts/s) (a) (b) θ ( 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, 2% and 3%. It was observed that higher percentage of chitosan resulted in the lower intensity in the X-ray diffraction patterns of the composites sample. 3% chitosan in the sample resulted in the lowering intensity of one fourth compared to the chitosan percentage of 2%. 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, 2% chitosan combined with 8% calcium phosphate compound and 3% chitosan combined with 7% calcium phosphate compound. Calcium phosphate ceramics consisted of HA and HA/CA mixture was also varied, 8% HA combined with 2% HA/CA mixture and 7% HA combined with 3% HA/CA mixture. This investigation presented that 2% 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. 2% chitosan was the most effective percentage.
6 Intensity (counts/s) International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 1 55 (a) (b ) o 2θ ( ) Figure 3 X-ray diffraction patterns of HA/chitosan composite with percentage of HA:chitosan of (a) (8:2)% and (b) (7:3)% It was also observed here that 2% HA/CA mixture combined with 8% 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 nm to nm for plain (2) and nm (a) (b) (c) Figure 4 SEM micrographs of calcium phosphate-chitosan composites with the percentage of HA:HACA:chitosan of (a) (64:16:2)%, (b) (56:24:2)% and (c) (:8:2)% IJENS
7 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol: 12 No: 1 56 to 2.91 nm for plain (3) by addition of 2%-3% chitosan in samples. For HA/CA samples, addition of chitosan apparently reduced the crystal size from 15.1 nm to nm for plain (2) and nm to 9.26 nm for plain (3). Surface morphology of chitosan showed small pores of about.1 to.2 µm in diameter, whilst surface morphology of HA showed crystals in the forms of bars of about.3 µm long. Surface morphology of HA/CA mixture showed collection of granules with the size of about 2 µm. It was not found that addition of 2% 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/2% chitosan composite showed granules with the size of about.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 8% 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 13 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. 2% 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 2% chitosan. References [1] C.T. Laurencin, A.M.A. Ambrosio, M.D. Borden, J.A. Cooper, Ann. Rev. Biomed. Eng. 1 (1999) 19. [2] R.D. Pilliar, M.J. Filiaggi, J.D. Wells, M.D. Grynpas, R.A. Kandel, Biomate. 22 (21) 963. [3] T.K. Daftari, T.R. Whitesides, J.G. Heller, Spine. 19 (1994) 94. [4] J.L. Meyer, E.D. Eanes, Calcif. Tiss. Ress. 25 (1978) 59. [5] S. Takagi, M. Mathew, J. Res. Natl. Stand. Technol. 16 (21) 13. [6] S. N. Danilchenko, A.V. Koropov, I. Yu. Protsenko, B. Sulkio-Cleff, L. F. Sukhodub, Cryst. Res. Technol. 41 (26) 268. [7] N. Pleshko, A. Boskey, R. Mendelsohn, Biophys. J. 6 (1991) 786. [8] S.N. Danilchenko, V.A. Pokrovskiy, V.M. Bogatyrov, L.F. Sukhodub, B. Sulkio-Cleff, Cryst. Res. Technol. 4 (25) 692. [9] L. Muller, F.A. Muller, A. Bioamate. 2 (26) 181.
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