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chondroitin sulfate composites by biomimetic synthesis
Materials Chemistry and Physics 112 (2008) 838–843
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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Preparation and characterization of hydroxyapatite/chondroitin sulfate composites by biomimetic synthesis Xiufeng Xiao, Dan He, Fang Liu, Rongfang Liu ∗ College of Chemistry and Materials Science, Fujian Normal University, Fujian, Fuzhou 350007, China
a r t i c l e
i n f o
Article history: Received 13 November 2007 Received in revised form 19 May 2008 Accepted 24 June 2008 Keywords: Biomaterials Nucleation Biomimetic Hydroxyapatite
a b s t r a c t Based on the principles of biomineralization, flakelike hydroxyapatite/chondroitin sulfate composites were synthesized through biomimetic method using Ca(NO3 )2 ·4H2 O and (NH4 )3 PO4 ·3H2 O as reagents and chondroitin sulfate as template. The crystalline phase, microstructure, chemical composition, morphology and thermal behavior of the composites obtained in the experiment were characterized by means of X-ray diffraction (XRD), Fourier transform infrared spectroscope (FTIR), transmission electron microscope (TEM), Thermogravimetry-Differential thermal analyzer (TG-DTA) and Elemental analyzer, respectively. The interaction between the functional groups of ChS and HA was investigated by electrical conductivity and UV–vis spectrum. The results demonstrate that the as-prepared powders with small amount of carbonate have the component similar to human bone. It can be concluded that the nucleation and growth of HA crystals occurred through the chemical interactions between the HA crystals and preorganized functional groups of the ChS template. Furthermore, the concentration of ChS significantly affects the morphology of the composites. Short fiberlike crystals could be obtained at a low concentration of ChS, but flakelike crystals could be synthesized using a high concentration (≥0.5 wt%) of ChS as template. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HA) is the main mineral constituent of natural bone and teeth, which has been widely used as the most promising biocompatible implant materials for orthopedic applications due to its excellent biocompatibility and bioactivity with bone replacement in living tissues [1]. Natural bone tissue is composed primarily of nano-size carbonated hydroxyapatite (CHA) crystals with the size of 40–60 nm in length, 10–20 nm in width and 1–3 nm in thickness [2]. So nano-size HA, which is more similar to the inorganic components in human body, has better biological properties. However, the poor mechanical properties of synthesized HA, such as high elastic modulus and low fracture toughness restrict its application. Thus, HA is used in the form of compound to retain useful bioactive properties as well as to enhance mechanical properties. In recent years, with the rapid development of the research on the nano-size HA materials, the molecular control of inorganic crystallization by organic substances is a key technology for the fabrication of novel inorganic/organic composites that has recently received a considerable amount of attention [3]. This process mimics biological mineralization in which a preorganized organic
∗ Corresponding author. Tel.: +86 591 83465190; fax: +86 591 87560183. E-mail address: rfl[email protected] (R. Liu). 0254-0584/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2008.06.055
phase provides a niche for inorganic crystals to nucleate and grow [4]. Nowadays, ethylene glycol [5], cetyltrimethyl ammonium bromide [6], polyethylene glycol [7], polyvinyl alcohol (PVA) [8], ethylenediamine tetraacetic acid (EDTA) [9], and mixed Tween80 with polyoxyethylene [10] are used as templates to synthesize HA. However, in nature, the nucleation and growth of mineralized materials are often controlled by organic macromolecules, such as proteins and polysaccharides. Chondroitin sulfate (ChS) is just one of the biomacromolecules. It is a kind of glycosaminoglycans (GAGs), which can be found on cell surfaces and in the extracellular matrix of cartilage and bone. A large amount of ChS in the cartilage permits the diffusion of substances between blood and vessels. ChS consists of repeated disaccharide units; one of the two monosaccharides is N-acetylgalactosamine sulfate (GalNAc-OSO3 − ), and the other is glucuronic acid that contains a carboxylate group (Fig. 1). The repeat length of per disaccharide is 0.913 nm and the molecular chain has three screw symmetry. The shape of unit cell is a trigonal prism with dimensions a = b = 1.28 nm, c = 2.74 nm, and = 120◦ [11]. ChS, the main GAG of tissue and proteoglycans (PGs), are present at the early stages of bone and tooth formation [12], but its role in the crystal growth process is not fully understood. Some studies show that ChS is an effective inhibitor of HA formation and growth from calcium phosphate solutions, as well as inhibitors of transformation of so-called amorphous calcium phosphate to crystalline HA [13,14]. The others show that the combination of sulfate
X. Xiao et al. / Materials Chemistry and Physics 112 (2008) 838–843
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phosphate ((NH4 )3 PO4 ·3H2 O, AR grade), and calcium nitrate (Ca(NO3 )2 ·4H2 O, AR grade) were from Chinese Medicine (Group) Company of Shanghai Chemical Reagents. 2.2. Synthesis of HA/ChS powder
Fig. 1. Schematic diagram of the structure of ChS.
and carboxylate groups gives ChS a very high density of negative charge and may let them to be nucleation sites for HA through binding the inorganic cations such as plus charged Ca ion [15,16]. The aim of the present study is to contribute to the understanding of the role of ChS in biomineralization processes. The initial intention is to synthesize pure nano-HA by biomimetic method using different concentrations of the ChS as templates. But from the results of elemental analysis and TG-DTA data, the obtained powders are the composites of HA/ChS. HA shows excellent biocompatibility with hard tissues, skin and muscle tissues [17]. And ChS is present at the early stages of bone and tooth formation [12]. So we can deduce that HA/ChS composites are biocompatible and the HA/ChS composites may be applicable to a biomimetic bone substitute [18]. 2. Experimental procedure
HA is a stoichiometric compound. Ca10 (PO4 )6 (OH)2 is often prepared at ambient temperature in our laboratory. 1.97 g Ca(NO3 )2 ·4H2 O and 1.02 g (NH4 )3 PO4 ·3H2 O were spread at the bottom of 100 and 25 ml beakers, respectively (keeping the molar ratio of Ca/P = 1.67). Then the smaller beaker was put into the bigger beaker. Fig. 2 shows the schematic diagram of experimental apparatus. The ChS solution with different concentrations (0.0, 0.1, 0.3, 0.5 and 0.7 mass%, respectively and the pH of the ChS solution adjusted to 11 using 1 mol L−1 NaOH solution) was added into the vessels along the inner beaker wall until the liquid phase was 5 mm higher than the inner beaker. Then the beakers were sealed with the preservative film and settled for fourteen days at room temperature. The obtained precipitates were washed with distilled water and ethanol three times, respectively. Then the precipitates were dried at 50 ◦ C overnight, ground in an agate mortar, and sealed in a dry bag. 2.3. Sample characterization The phase composition of as-prepared powders was determined using X-ray diffraction (XRD). A Philips X’Pert MPD diffractometer with Cu Ka radiation was used, and the X-ray generator operated at 40 KV and 40 mA. Data sets were collected over the range from 5◦ to 90◦ with a step size of 0.02◦ and a count rate of 3.0◦ min−1 . Phase identification was achieved by comparing the diffraction patterns of HA with ICDD standards (JCPDS 09-432). Fourier transform infrared spectra (FTIR) were obtained using Nicolet Avatar 360 spectrometer at 4 cm−1 resolution averaging 64 scans. A Hitachi 600 transmission electron microscope (TEM) was employed for characterization of the morphology of the samples. The thermal behavior of the HA powder was determined by TG-DTA (Nicolet 5700 FTIR Mettler TGA/SDTA 851) at a heating rate of 3 ◦ C min−1 to 900 ◦ C in an N2 atmosphere. The electrical conductivity was measured via a DDS-307A conductometer at 25 ◦ C.
2.1. Materials Chondroitin sulfate was purchased from Shandong liangshan kejing Bioengineering Co. Ltd. (BR grade). Absolute ethanol (C2 H5 OH, AR grade), ammonium
3. Results and discussion 3.1. XRD analyses XRD patterns of as-prepared powders in pure water and using different concentrations of the ChS as templates are shown in Fig. 3. The crystal diffraction peaks observed at 2 = 26◦ and 32◦ of the samples synthesized are the same as the standard HA diffraction peaks (JCPDS 09-432) without the other phase detected. However, the diffraction peaks of the sample obtained in the absence of ChS (Fig. 3a) are less intense than that in the presence of ChS (Fig. 3b–e). This suggests that the samples are HA crystals in the presence of very small crystallites. From the full width at half maximum (FWHM), the average crystalline size can be estimated with the (0 0 2) (2 = 25.8◦ ) diffraction
Fig. 2. The chart of experimental apparatus.
Fig. 3. XRD patterns of the samples obtained with different concentrations of ChS as templates. (a) The absence of ChS, ChS: (b) 0.1 wt%, (c) 0.3 wt%, (d) 0.5 wt%, (e) 0.7 wt%.
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Table 1 The grain size of obtained samples with the existence of different concentrations of the ChS
Table 2 Elemental analyses data of ChS and as-prepared sample using 0.7 wt%ChS as templates
Concentration of ChS (%)
FWHM
The crystallite size (D) nm
Sample
C%
H%
N%
0.0 0.1 0.3 0.5 0.7
0.3346 0.3011 0.2896 0.2767 0.2676
25.8728 28.7513 29.8930 31.2867 32.3506
ChS Sample
30.67 4.645
5.384 1.665
3.482 0.530
peaks in the XRD patterns according to the Scherrer formula [19]: D=
k ˇ cos
D is the crystallite size(nm); K = 0.89, which is the Scherrer approximate constant; is the wavelength of the X-ray (Cu K␣, 0.15406 nm); is the diffraction angle (◦ ); and ˇ is the experimental full width at half maximum (FWHM). The effect of geometric (instrumental) broadening on the diffraction peaks was calibrated. The results are shown in Table 1. The crystal size increases as the concentration of ChS increases, indicating larger crystallites and higher crystallinity. So it could be concluded that ChS has effects on the nucleation and growth of HA. 3.2. FTIR analyses The FTIR spectra of pure ChS and the samples obtained with different concentrations of ChS as templates are shown in Fig. 4. Fig. 4a shows the characteristic absorption bands of ChS, the asymmetrical stretching model of N H, COO− , SO3 − and C4 O S are detected at around 3421, 1632, 1240 and 851 cm−1 , respectively [20]. Fig. 4b–f shows the characteristic absorption bands of HA/ChS composites. The bands at 1044, 604 and 558 cm−1 are attributed to PO4 3− group. With the increasing concentration of ChS, the three PO4 3− peaks (1044, 604 and 558 cm−1 ) become stronger. These changes suggest that the crystallinity of the apatite increases as the concentration of ChS increases, which agrees with the results of the XRD patterns. That is to say, the presence of ChS can accelerate the nucleation and growth of HA crystals. The shoulder bands at 3571 and 631 cm−1 correspond to the stretching and vibrational modes, respectively, of OH− group. The peaks at 872, 1430 and 1450 cm−1 are due to the stretching and bending modes of CO3 2− ions, which are derived from the absorption of CO2 in the air during preparation
process of HA [21]. It means that PO4 3− sites of the HA structure, i.e. B-site, were partly substituted by carbonate ions [22]. And the peaks at 1240 cm−1 are ascribed to the asymmetrical stretching model of SO3 − of ChS. The N H, COO− and − C4 O S of ChS overlap with the absorption bands of the characteristic of H2 O (3445 and 1650 cm−1 ) and CO3 2− (872 cm−1 ) of HA. So the very broad band at 3445 and 1650 cm−1 can be seen in all samples. 3.3. Morphologies of the samples The TEM micrographs of as-prepared powders are shown in Fig. 5. Fig. 5 shows that the obtained samples agglomerate seriously in a flocculent morphology without ChS templates (Fig. 5a) or with the small amount of ChS as templates (Fig. 5b). When the concentration of ChS is increased to 0.3 wt%, the dispersity of the sample can be improved and the agglomerate state of the sample can be reduced. Furthermore, the morphology of some granules changes from flocculentlike crystals to fiberlike crystals (Fig. 5c). The samples appear in a flakelike structure when the concentration of ChS is increased to 0.5 wt%, but the particle size of the sample is not uniform, and the edges of the particles are not very smooth (Fig. 5d). All particles become flakelike crystals with the size of 100 nm in length and 40 nm in width (Fig. 5e) when the concentration of ChS is relative high (0.7 wt%). It can be concluded that ChS molecule has an important effect on the growth behavior and the morphology of HA. 3.4. Elemental analyses The elemental analyses shown in Table 2 indicate that the sample synthesized with ChS as templates contain small amount of ChS. According to the content of N Elementary in ChS and in the sample synthesized with 0.7 wt%ChS as templates, the content of ChS, about 15.46 wt%, in the sample can be calculated. ChS is a water-soluble compound, the sample has been washed with distilled water three times, but ChS is still there in the sample, which further demonstrates the interaction between ChS and HA, and the sample can be considered to be a HA/ChS complex. 3.5. TG-DTA analyses TG-DTA curves of the obtained sample with 0.7 wt%ChS as templates are shown in Fig. 6. The TG curve shows a total weight loss of 21.9 wt% at 900 ◦ C. The wide endothermic peaks between 60 and 650 ◦ C observed in the DTA curves are attributed to the evaporation of the absorbed water of HA and the decomposition of a little ChS remained in powders accompanied by significant weight loss. The corresponding HA dehydration endothermic peak occurs at 700 ◦ C. Therefore, it leads to the conclusion that the powders synthesized with ChS as templates contain ChS. 3.6. Formation mechanism
Fig. 4. FTIR spectra of the samples obtained with different concentrations of ChS as templates. (a) Pure ChS, (b) The absence of ChS, ChS: (c) 0.1 wt%, (d) 0.3 wt%, (e) 0.5 wt%, (f) 0.7 wt%.
The investigation of electrical conductivity is an important method to study the interaction between Ca(NO3 )2 and ChS. Electrical conductivity of different concentrations of Ca(NO3 )2 solution and corresponding Ca(NO3 )2 –0.5 wt%ChS solution is
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Fig. 5. TEM micrographs of the samples obtained with different concentrations of ChS as templates. (a) The absence of ChS; ChS: (b) 0.1 wt%, (C) 0.3 wt%, (d) 0.5 wt%, (e) 0.7 wt%.
shown in Table 3. It can be seen that electrical conductivity of Ca(NO3 )2 –ChS solution is lower than the sum total electrical conductivity of Ca(NO3 )2 and 0.5 wt%ChS. The difference of electrical conductivity(S) increases from 0.41 to 1.02 ms cm−1 as the concentration of Ca(NO3 )2 increases. This proves that there is inter-
action between Ca2+ and ChS. Electrical conductivity of the solution is affected by the ions concentration and ions velocity. The function groups COO− and SO3 − in ChS can be bound by Ca2+ ions in alkaline medium, which reduce the drifting velocity and the concentration of ions, resulting in the decrease of electrical conductivity.
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Fig. 7. Ultraviolet absorption spectra of the solution. (a) 0.5 wt%ChS solution, (b) Ca(NO3 )2 –0.5 wt%ChS solution. Fig. 6. TG-DTA curves of the as-prepared sample with 0.7% ChS as templates.
Table 3 Electrical conductivity of Ca(NO3 )2 and Ca(NO3 )2 –ChS solution (25 ◦ C) C(Ca(NO3 )2 ) (mol L−1 )
Electrical conductivity
S1 (Ca(NO3 )2 + 0.5%ChS) (ms cm−1 ) S2 (Ca(NO3 )2 – ChS) (ms cm−1 ) S = S1 − S2
0.02
0.04
0.06
0.08
0.10
4.89 4.48 0.41
8.54 7.92 0.62
11.89 11.18 0.71
15.03 14.20 0.83
17.87 16.85 1.02
Ultraviolet absorption spectra of the 0.5 wt%ChS solution and Ca(NO3 )2 –0.5 wt%ChS solution are shown in Fig. 7. The absorption peaks in 219 and 267 nm (Fig. 7a) are ascribed to COO− of ChS. When Ca2+ ion was added into ChS solution, the absorption peak’s blue shift to 234 and 300 nm (Fig. 7b), respectively and the absorption peak intensity of COO− decreases. This is probably a result of the combination between the negatively charged COO− and the positively charged Ca2+ ions. After binding between COO− , SO3 − and Ca2+ , PO4 3− and OH− ions will move to these Ca2+ ions by electrostatic attraction, which
increases the local supersaturation of HA and induces the nucleation and growth of HA with ChS as templates. This interaction may be one of the reasons that influence the nucleation and growth of HA. Therefore, FTIR and XRD results indicate that the existence of ChS is in favor of the formation of HA, and the crystallinity of HA increases as concentration of ChS increases. The effect of ChS on the morphology of HA may be related to structure of ChS (Fig. 1). ChS is a linear polysaccharide of repeated disaccharide units. Such a unit contains sulfate groups and carboxylate groups. Upon ionization, the negatively charged surface groups can provide binding sites for the Ca ions and locally produces a high degree of supersaturation in the solvent. As the calcium ions accumulate on the surface, the surface gains an overall positive charge. This process is followed by the attraction of negatively charged phosphate ions PO4 3− and OH− towards these uniformly distributed Ca sites, initiating the crystallization process of HA on ChS (Fig. 8a). Such an electrostatic model for the hydroxyapatite formation has been indicated in earlier studies [23–28].The binding between calcium ions and the charged groups in the polymer phase at this molecular level has been shown to
Fig. 8. Schematic diagram for the crystallochemical specific nucleation and growth of the HA crystals on the ChS templates.
X. Xiao et al. / Materials Chemistry and Physics 112 (2008) 838–843
have a positive effect on the mechanical behavior of the composite systems prepared in this manner [26]. Due to the interaction between the HA crystals and the functional groups of the ChS, the crystallographic c-axis of the HA crystals are preferentially aligned to parallel to the long axis direction of the ChS templates [18,20]. So the fiberlike HA was obtained at a low concentration of ChS (Fig. 8b). But when the concentration of ChS increases to 0.5 and 0.7 wt% in the solution, many ChS chains mutually crosslink, then lead to the formation of a two-dimensional network structure. Ca2+ , PO4 3− and OH− nucleate and grow on the surface of ChS by the above-mentioned electrostatic matching and geometry matching. So flakelike HA was obtained. The higher the concentration of ChS, the more the ChS chains crosslink [29]. Consequently, the bigger flakelike HA obtained (Fig. 8c). 4. Conclusion HA/ChS composites were prepared by biomimetic method with ChS as templates. The formation of the composites at room temperature was confirmed by both the XRD patterns and the presence of characteristic absorption bands in the FTIR spectra. The results show that the existence of ChS is in favor of the formation of HA, and the crystallinity of HA increases as the concentration of ChS increases. TEM analyses indicate that ChS molecule has an important effect on the growth behavior and the morphology of HA. The elemental analyses and TG-DTA data point out that the obtained powders are the formation of a HA/ChS complex. Furthermore, the shifts in the maximum of the absorption band of Ca(NO3 )2 –ChS in ultraviolet absorption spectra and the change of electrical conductivity of Ca(NO3 )2 and Ca(NO3 )2 –ChS prove that there is an interaction between Ca2+ and ChS, and this interaction may be one of the reasons which influence the nucleation and growth of HA. Acknowledgements The authors would like to thank National Nature Science Foundation of China (30600149), the science research foundation of
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ministry of Health & United Fujian Provincial Health and Education Project for Tackling the Key Research, P.R. China (WKJ 2005-2008), Fujian Development and Reform Commission of China (No. 2004[477]) and Fujian Provincial Department of Science and Technology (No. 2006I0015). References [1] K.C.B. Yeong, J. Wang, S.C. Ng, Biomaterials 22 (2001) 2705. [2] X.M. Deng, J.Y. Hao, C.S. Wang, Biomaterials 22 (2001) 2867. [3] S. Mann, D.D. Archibald, J.M. Didymus, T. Douglas, B.R. Heywood, F.C. Meldrum, N.J. Reeves, Science 261 (1993) 1286. [4] B.C. Bunker, P.C. Rieke, B.J. Tarasevich, A.A. Campbell, G.E. Fryxell, G.L. Graff, L. Song, J. Liu, W. Virden, G.L. Mcvay, Science 264 (1994) 48. [5] A. Milev, G.S.K. Kannangara, B. Ben-Nissan, Mater. Lett. 57 (2003) 1960. [6] Y. Liu, W. Wang, Y. Zhan, C. Zheng, G. Wang, Mater. Lett. 56 (2002) 496. [7] A.L. Wang, H.B. Yin, D. Liu a, H.X. Wu, Y. Wada, M. Ren, Y.Q. Xu, T.S. Jiang, X.N. Cheng, Appl. Surf. Sci. 253 (2007) 3311. [8] L. Yan, Y. Li, Z.X. Deng, J. Zhuang, X. Sun, Int. J. Inorg. Mater. 3 (2001) 633. [9] J. Liu, K. Li, H. Wang, M. Zhu, H. Yan, Chem. Phys. Lett. 396 (2004) 429. [10] G. Guo, Y. Sun, Z. Wang, H. Guo, Ceram. Int. 31 (2005) 869. [11] K. Tanaka, J. Biochem. 83 (1978) 647. [12] M. Goldberg, O. Rapoport, D. Septier, K. Palmier, R. Hall, G. Embery, M. Young, L. Ameye, Connect. Tissue Res. 44 (2003) 184. [13] D.S. Howell, J.C. Pita, Clin. Orthop. 118 (1976) 208. [14] C.C. Chen, A.L. Boskey, L.C. Rosenberg, Calcif. Tissue Int. 36 (1984) 285. [15] L. Kjellen, U. Lindahl, Ann. Rev. Biochem. 60 (1991) 443. [16] G.K. Hunter, S.M. Bader, J. Theor. Biol. 138 (1989) 195. [17] Y.F. Hu, X.G. Miao, Ceram. Int. 30 (2004) 1787. [18] S.H. Rhee, Y. Suetsugu, J. Tanaka, Biomaterials 22 (2001) 2843. [19] Z.Q. Wei, T.D. Xia, W.J. Feng, J.F. Dai, Q. Wang, W.X. Li, P.X. Yan, Raremetals 25 (2006) 172. [20] S.H. Rhee, J. Tanaka, J. Mater. Sci: Mater. Med. 13 (2002) 597. [21] S. Mollazadeh, J. Javadpour, A. Khavandi, Ceram. Int. 33 (2007) 1579. [22] Y. Suetsugu, I. Shimoya, J. Tanaka, J. Am. Geram. Soc. 81 (1998) 746. [23] M. Kikuclin, S. Itoh, S. Ichinose, K. Shinomiya, J. Tanaka, Biomaterials 22 (2001) 1705. [24] G.K. Toworfe, R.J. Composto, I.M. Shapiro, P. Ducheyne, Biomaterials 27 (2006) 631. [25] X. Wang, Y. Li, J. Wei, K. de Groot, Biomaterials 23 (2002) 4787. [26] T. Kokubo, H.M. Kim, M. Kawashata, Biomaterials 24 (2004) 2161. [27] E. Dalas, A. Chrissanthopoulos, J. Cryst. Growth 255 (2003) 163. [28] P. Zhu, Y. Masuda, K. Koumoto, Biomaterials 25 (2004) 3915. [29] K. Xia, J.Y. Cai, Pharm. Biotechnol. 10 (2003) 10–13.
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Preparation and characterization of hydroxyapatite/chondroitin sulfate composites by biomimetic synthesis Xiufeng Xiao, Dan He, Fang Liu, Rongfang Liu ∗ College of Chemistry and Materials Science, Fujian Normal University, Fujian, Fuzhou 350007, China
a r t i c l e
i n f o
Article history: Received 13 November 2007 Received in revised form 19 May 2008 Accepted 24 June 2008 Keywords: Biomaterials Nucleation Biomimetic Hydroxyapatite
a b s t r a c t Based on the principles of biomineralization, flakelike hydroxyapatite/chondroitin sulfate composites were synthesized through biomimetic method using Ca(NO3 )2 ·4H2 O and (NH4 )3 PO4 ·3H2 O as reagents and chondroitin sulfate as template. The crystalline phase, microstructure, chemical composition, morphology and thermal behavior of the composites obtained in the experiment were characterized by means of X-ray diffraction (XRD), Fourier transform infrared spectroscope (FTIR), transmission electron microscope (TEM), Thermogravimetry-Differential thermal analyzer (TG-DTA) and Elemental analyzer, respectively. The interaction between the functional groups of ChS and HA was investigated by electrical conductivity and UV–vis spectrum. The results demonstrate that the as-prepared powders with small amount of carbonate have the component similar to human bone. It can be concluded that the nucleation and growth of HA crystals occurred through the chemical interactions between the HA crystals and preorganized functional groups of the ChS template. Furthermore, the concentration of ChS significantly affects the morphology of the composites. Short fiberlike crystals could be obtained at a low concentration of ChS, but flakelike crystals could be synthesized using a high concentration (≥0.5 wt%) of ChS as template. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HA) is the main mineral constituent of natural bone and teeth, which has been widely used as the most promising biocompatible implant materials for orthopedic applications due to its excellent biocompatibility and bioactivity with bone replacement in living tissues [1]. Natural bone tissue is composed primarily of nano-size carbonated hydroxyapatite (CHA) crystals with the size of 40–60 nm in length, 10–20 nm in width and 1–3 nm in thickness [2]. So nano-size HA, which is more similar to the inorganic components in human body, has better biological properties. However, the poor mechanical properties of synthesized HA, such as high elastic modulus and low fracture toughness restrict its application. Thus, HA is used in the form of compound to retain useful bioactive properties as well as to enhance mechanical properties. In recent years, with the rapid development of the research on the nano-size HA materials, the molecular control of inorganic crystallization by organic substances is a key technology for the fabrication of novel inorganic/organic composites that has recently received a considerable amount of attention [3]. This process mimics biological mineralization in which a preorganized organic
∗ Corresponding author. Tel.: +86 591 83465190; fax: +86 591 87560183. E-mail address: rfl[email protected] (R. Liu). 0254-0584/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2008.06.055
phase provides a niche for inorganic crystals to nucleate and grow [4]. Nowadays, ethylene glycol [5], cetyltrimethyl ammonium bromide [6], polyethylene glycol [7], polyvinyl alcohol (PVA) [8], ethylenediamine tetraacetic acid (EDTA) [9], and mixed Tween80 with polyoxyethylene [10] are used as templates to synthesize HA. However, in nature, the nucleation and growth of mineralized materials are often controlled by organic macromolecules, such as proteins and polysaccharides. Chondroitin sulfate (ChS) is just one of the biomacromolecules. It is a kind of glycosaminoglycans (GAGs), which can be found on cell surfaces and in the extracellular matrix of cartilage and bone. A large amount of ChS in the cartilage permits the diffusion of substances between blood and vessels. ChS consists of repeated disaccharide units; one of the two monosaccharides is N-acetylgalactosamine sulfate (GalNAc-OSO3 − ), and the other is glucuronic acid that contains a carboxylate group (Fig. 1). The repeat length of per disaccharide is 0.913 nm and the molecular chain has three screw symmetry. The shape of unit cell is a trigonal prism with dimensions a = b = 1.28 nm, c = 2.74 nm, and = 120◦ [11]. ChS, the main GAG of tissue and proteoglycans (PGs), are present at the early stages of bone and tooth formation [12], but its role in the crystal growth process is not fully understood. Some studies show that ChS is an effective inhibitor of HA formation and growth from calcium phosphate solutions, as well as inhibitors of transformation of so-called amorphous calcium phosphate to crystalline HA [13,14]. The others show that the combination of sulfate
X. Xiao et al. / Materials Chemistry and Physics 112 (2008) 838–843
839
phosphate ((NH4 )3 PO4 ·3H2 O, AR grade), and calcium nitrate (Ca(NO3 )2 ·4H2 O, AR grade) were from Chinese Medicine (Group) Company of Shanghai Chemical Reagents. 2.2. Synthesis of HA/ChS powder
Fig. 1. Schematic diagram of the structure of ChS.
and carboxylate groups gives ChS a very high density of negative charge and may let them to be nucleation sites for HA through binding the inorganic cations such as plus charged Ca ion [15,16]. The aim of the present study is to contribute to the understanding of the role of ChS in biomineralization processes. The initial intention is to synthesize pure nano-HA by biomimetic method using different concentrations of the ChS as templates. But from the results of elemental analysis and TG-DTA data, the obtained powders are the composites of HA/ChS. HA shows excellent biocompatibility with hard tissues, skin and muscle tissues [17]. And ChS is present at the early stages of bone and tooth formation [12]. So we can deduce that HA/ChS composites are biocompatible and the HA/ChS composites may be applicable to a biomimetic bone substitute [18]. 2. Experimental procedure
HA is a stoichiometric compound. Ca10 (PO4 )6 (OH)2 is often prepared at ambient temperature in our laboratory. 1.97 g Ca(NO3 )2 ·4H2 O and 1.02 g (NH4 )3 PO4 ·3H2 O were spread at the bottom of 100 and 25 ml beakers, respectively (keeping the molar ratio of Ca/P = 1.67). Then the smaller beaker was put into the bigger beaker. Fig. 2 shows the schematic diagram of experimental apparatus. The ChS solution with different concentrations (0.0, 0.1, 0.3, 0.5 and 0.7 mass%, respectively and the pH of the ChS solution adjusted to 11 using 1 mol L−1 NaOH solution) was added into the vessels along the inner beaker wall until the liquid phase was 5 mm higher than the inner beaker. Then the beakers were sealed with the preservative film and settled for fourteen days at room temperature. The obtained precipitates were washed with distilled water and ethanol three times, respectively. Then the precipitates were dried at 50 ◦ C overnight, ground in an agate mortar, and sealed in a dry bag. 2.3. Sample characterization The phase composition of as-prepared powders was determined using X-ray diffraction (XRD). A Philips X’Pert MPD diffractometer with Cu Ka radiation was used, and the X-ray generator operated at 40 KV and 40 mA. Data sets were collected over the range from 5◦ to 90◦ with a step size of 0.02◦ and a count rate of 3.0◦ min−1 . Phase identification was achieved by comparing the diffraction patterns of HA with ICDD standards (JCPDS 09-432). Fourier transform infrared spectra (FTIR) were obtained using Nicolet Avatar 360 spectrometer at 4 cm−1 resolution averaging 64 scans. A Hitachi 600 transmission electron microscope (TEM) was employed for characterization of the morphology of the samples. The thermal behavior of the HA powder was determined by TG-DTA (Nicolet 5700 FTIR Mettler TGA/SDTA 851) at a heating rate of 3 ◦ C min−1 to 900 ◦ C in an N2 atmosphere. The electrical conductivity was measured via a DDS-307A conductometer at 25 ◦ C.
2.1. Materials Chondroitin sulfate was purchased from Shandong liangshan kejing Bioengineering Co. Ltd. (BR grade). Absolute ethanol (C2 H5 OH, AR grade), ammonium
3. Results and discussion 3.1. XRD analyses XRD patterns of as-prepared powders in pure water and using different concentrations of the ChS as templates are shown in Fig. 3. The crystal diffraction peaks observed at 2 = 26◦ and 32◦ of the samples synthesized are the same as the standard HA diffraction peaks (JCPDS 09-432) without the other phase detected. However, the diffraction peaks of the sample obtained in the absence of ChS (Fig. 3a) are less intense than that in the presence of ChS (Fig. 3b–e). This suggests that the samples are HA crystals in the presence of very small crystallites. From the full width at half maximum (FWHM), the average crystalline size can be estimated with the (0 0 2) (2 = 25.8◦ ) diffraction
Fig. 2. The chart of experimental apparatus.
Fig. 3. XRD patterns of the samples obtained with different concentrations of ChS as templates. (a) The absence of ChS, ChS: (b) 0.1 wt%, (c) 0.3 wt%, (d) 0.5 wt%, (e) 0.7 wt%.
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Table 1 The grain size of obtained samples with the existence of different concentrations of the ChS
Table 2 Elemental analyses data of ChS and as-prepared sample using 0.7 wt%ChS as templates
Concentration of ChS (%)
FWHM
The crystallite size (D) nm
Sample
C%
H%
N%
0.0 0.1 0.3 0.5 0.7
0.3346 0.3011 0.2896 0.2767 0.2676
25.8728 28.7513 29.8930 31.2867 32.3506
ChS Sample
30.67 4.645
5.384 1.665
3.482 0.530
peaks in the XRD patterns according to the Scherrer formula [19]: D=
k ˇ cos
D is the crystallite size(nm); K = 0.89, which is the Scherrer approximate constant; is the wavelength of the X-ray (Cu K␣, 0.15406 nm); is the diffraction angle (◦ ); and ˇ is the experimental full width at half maximum (FWHM). The effect of geometric (instrumental) broadening on the diffraction peaks was calibrated. The results are shown in Table 1. The crystal size increases as the concentration of ChS increases, indicating larger crystallites and higher crystallinity. So it could be concluded that ChS has effects on the nucleation and growth of HA. 3.2. FTIR analyses The FTIR spectra of pure ChS and the samples obtained with different concentrations of ChS as templates are shown in Fig. 4. Fig. 4a shows the characteristic absorption bands of ChS, the asymmetrical stretching model of N H, COO− , SO3 − and C4 O S are detected at around 3421, 1632, 1240 and 851 cm−1 , respectively [20]. Fig. 4b–f shows the characteristic absorption bands of HA/ChS composites. The bands at 1044, 604 and 558 cm−1 are attributed to PO4 3− group. With the increasing concentration of ChS, the three PO4 3− peaks (1044, 604 and 558 cm−1 ) become stronger. These changes suggest that the crystallinity of the apatite increases as the concentration of ChS increases, which agrees with the results of the XRD patterns. That is to say, the presence of ChS can accelerate the nucleation and growth of HA crystals. The shoulder bands at 3571 and 631 cm−1 correspond to the stretching and vibrational modes, respectively, of OH− group. The peaks at 872, 1430 and 1450 cm−1 are due to the stretching and bending modes of CO3 2− ions, which are derived from the absorption of CO2 in the air during preparation
process of HA [21]. It means that PO4 3− sites of the HA structure, i.e. B-site, were partly substituted by carbonate ions [22]. And the peaks at 1240 cm−1 are ascribed to the asymmetrical stretching model of SO3 − of ChS. The N H, COO− and − C4 O S of ChS overlap with the absorption bands of the characteristic of H2 O (3445 and 1650 cm−1 ) and CO3 2− (872 cm−1 ) of HA. So the very broad band at 3445 and 1650 cm−1 can be seen in all samples. 3.3. Morphologies of the samples The TEM micrographs of as-prepared powders are shown in Fig. 5. Fig. 5 shows that the obtained samples agglomerate seriously in a flocculent morphology without ChS templates (Fig. 5a) or with the small amount of ChS as templates (Fig. 5b). When the concentration of ChS is increased to 0.3 wt%, the dispersity of the sample can be improved and the agglomerate state of the sample can be reduced. Furthermore, the morphology of some granules changes from flocculentlike crystals to fiberlike crystals (Fig. 5c). The samples appear in a flakelike structure when the concentration of ChS is increased to 0.5 wt%, but the particle size of the sample is not uniform, and the edges of the particles are not very smooth (Fig. 5d). All particles become flakelike crystals with the size of 100 nm in length and 40 nm in width (Fig. 5e) when the concentration of ChS is relative high (0.7 wt%). It can be concluded that ChS molecule has an important effect on the growth behavior and the morphology of HA. 3.4. Elemental analyses The elemental analyses shown in Table 2 indicate that the sample synthesized with ChS as templates contain small amount of ChS. According to the content of N Elementary in ChS and in the sample synthesized with 0.7 wt%ChS as templates, the content of ChS, about 15.46 wt%, in the sample can be calculated. ChS is a water-soluble compound, the sample has been washed with distilled water three times, but ChS is still there in the sample, which further demonstrates the interaction between ChS and HA, and the sample can be considered to be a HA/ChS complex. 3.5. TG-DTA analyses TG-DTA curves of the obtained sample with 0.7 wt%ChS as templates are shown in Fig. 6. The TG curve shows a total weight loss of 21.9 wt% at 900 ◦ C. The wide endothermic peaks between 60 and 650 ◦ C observed in the DTA curves are attributed to the evaporation of the absorbed water of HA and the decomposition of a little ChS remained in powders accompanied by significant weight loss. The corresponding HA dehydration endothermic peak occurs at 700 ◦ C. Therefore, it leads to the conclusion that the powders synthesized with ChS as templates contain ChS. 3.6. Formation mechanism
Fig. 4. FTIR spectra of the samples obtained with different concentrations of ChS as templates. (a) Pure ChS, (b) The absence of ChS, ChS: (c) 0.1 wt%, (d) 0.3 wt%, (e) 0.5 wt%, (f) 0.7 wt%.
The investigation of electrical conductivity is an important method to study the interaction between Ca(NO3 )2 and ChS. Electrical conductivity of different concentrations of Ca(NO3 )2 solution and corresponding Ca(NO3 )2 –0.5 wt%ChS solution is
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Fig. 5. TEM micrographs of the samples obtained with different concentrations of ChS as templates. (a) The absence of ChS; ChS: (b) 0.1 wt%, (C) 0.3 wt%, (d) 0.5 wt%, (e) 0.7 wt%.
shown in Table 3. It can be seen that electrical conductivity of Ca(NO3 )2 –ChS solution is lower than the sum total electrical conductivity of Ca(NO3 )2 and 0.5 wt%ChS. The difference of electrical conductivity(S) increases from 0.41 to 1.02 ms cm−1 as the concentration of Ca(NO3 )2 increases. This proves that there is inter-
action between Ca2+ and ChS. Electrical conductivity of the solution is affected by the ions concentration and ions velocity. The function groups COO− and SO3 − in ChS can be bound by Ca2+ ions in alkaline medium, which reduce the drifting velocity and the concentration of ions, resulting in the decrease of electrical conductivity.
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Fig. 7. Ultraviolet absorption spectra of the solution. (a) 0.5 wt%ChS solution, (b) Ca(NO3 )2 –0.5 wt%ChS solution. Fig. 6. TG-DTA curves of the as-prepared sample with 0.7% ChS as templates.
Table 3 Electrical conductivity of Ca(NO3 )2 and Ca(NO3 )2 –ChS solution (25 ◦ C) C(Ca(NO3 )2 ) (mol L−1 )
Electrical conductivity
S1 (Ca(NO3 )2 + 0.5%ChS) (ms cm−1 ) S2 (Ca(NO3 )2 – ChS) (ms cm−1 ) S = S1 − S2
0.02
0.04
0.06
0.08
0.10
4.89 4.48 0.41
8.54 7.92 0.62
11.89 11.18 0.71
15.03 14.20 0.83
17.87 16.85 1.02
Ultraviolet absorption spectra of the 0.5 wt%ChS solution and Ca(NO3 )2 –0.5 wt%ChS solution are shown in Fig. 7. The absorption peaks in 219 and 267 nm (Fig. 7a) are ascribed to COO− of ChS. When Ca2+ ion was added into ChS solution, the absorption peak’s blue shift to 234 and 300 nm (Fig. 7b), respectively and the absorption peak intensity of COO− decreases. This is probably a result of the combination between the negatively charged COO− and the positively charged Ca2+ ions. After binding between COO− , SO3 − and Ca2+ , PO4 3− and OH− ions will move to these Ca2+ ions by electrostatic attraction, which
increases the local supersaturation of HA and induces the nucleation and growth of HA with ChS as templates. This interaction may be one of the reasons that influence the nucleation and growth of HA. Therefore, FTIR and XRD results indicate that the existence of ChS is in favor of the formation of HA, and the crystallinity of HA increases as concentration of ChS increases. The effect of ChS on the morphology of HA may be related to structure of ChS (Fig. 1). ChS is a linear polysaccharide of repeated disaccharide units. Such a unit contains sulfate groups and carboxylate groups. Upon ionization, the negatively charged surface groups can provide binding sites for the Ca ions and locally produces a high degree of supersaturation in the solvent. As the calcium ions accumulate on the surface, the surface gains an overall positive charge. This process is followed by the attraction of negatively charged phosphate ions PO4 3− and OH− towards these uniformly distributed Ca sites, initiating the crystallization process of HA on ChS (Fig. 8a). Such an electrostatic model for the hydroxyapatite formation has been indicated in earlier studies [23–28].The binding between calcium ions and the charged groups in the polymer phase at this molecular level has been shown to
Fig. 8. Schematic diagram for the crystallochemical specific nucleation and growth of the HA crystals on the ChS templates.
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have a positive effect on the mechanical behavior of the composite systems prepared in this manner [26]. Due to the interaction between the HA crystals and the functional groups of the ChS, the crystallographic c-axis of the HA crystals are preferentially aligned to parallel to the long axis direction of the ChS templates [18,20]. So the fiberlike HA was obtained at a low concentration of ChS (Fig. 8b). But when the concentration of ChS increases to 0.5 and 0.7 wt% in the solution, many ChS chains mutually crosslink, then lead to the formation of a two-dimensional network structure. Ca2+ , PO4 3− and OH− nucleate and grow on the surface of ChS by the above-mentioned electrostatic matching and geometry matching. So flakelike HA was obtained. The higher the concentration of ChS, the more the ChS chains crosslink [29]. Consequently, the bigger flakelike HA obtained (Fig. 8c). 4. Conclusion HA/ChS composites were prepared by biomimetic method with ChS as templates. The formation of the composites at room temperature was confirmed by both the XRD patterns and the presence of characteristic absorption bands in the FTIR spectra. The results show that the existence of ChS is in favor of the formation of HA, and the crystallinity of HA increases as the concentration of ChS increases. TEM analyses indicate that ChS molecule has an important effect on the growth behavior and the morphology of HA. The elemental analyses and TG-DTA data point out that the obtained powders are the formation of a HA/ChS complex. Furthermore, the shifts in the maximum of the absorption band of Ca(NO3 )2 –ChS in ultraviolet absorption spectra and the change of electrical conductivity of Ca(NO3 )2 and Ca(NO3 )2 –ChS prove that there is an interaction between Ca2+ and ChS, and this interaction may be one of the reasons which influence the nucleation and growth of HA. Acknowledgements The authors would like to thank National Nature Science Foundation of China (30600149), the science research foundation of
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