S a A a b c a A R R 1 A A K B C M 1 a p s e w i a t t c b d t c 0 d Applied Surface Science 257 (2011) 3888–3892 Contents lists available at ScienceDirect Applied Surface Science journa l homepage: www.e lsev ier .com/ locate /apsusc ynthesis of chitosan/hydroxyapatite membranes coated with hydroxycarbonate patite for guided tissue regeneration purposes lexandre Félix Fragaa, Edson de Almeida Filhoc,∗, Eliana Cristina da Silva Rigob, Anselmo Ortega Boschia Federal University of São Carlos, Department of Materials Engineering, DEMa, UFSCar, São Carlos, SP, Brazil University of São Paulo, Department of Basic Science – FZEA-ZAB, Pirassununga, SP, Brazil University Estadual Paulista, Department of Physical Chemistry – IQ, Araraquara, SP, Brazil r t i c l e i n f o rticle history: eceived 8 July 2010 eceived in revised form 6 November 2010 ccepted 16 November 2010 vailable online 9 December 2010 eywords: a b s t r a c t Chitosan, which is a non-toxic, biodegradable and biocompatible biopolymer, has been widely researched for several applications in the field of biomaterials. Calcium phosphate ceramics stand out among the so-called bioceramics for their absence of local or systemic toxicity, their non-response to foreign bodies or inflammations, and their apparent ability to bond to the host tissue. Hydroxyapatite (HA) is one of the most important bioceramics because it is the main component of the mineral phase of bone. The aim of this work was to produce chitosan membranes coated with hydroxyapatite using the modified biomimetic method. Membranes were synthesized from a solution containing 2% of chitosan in acetic iomimetic method hitosan embranes and hydroxyapatite acid (weight/volume) via the solvent evaporation method. Specimens were immersed in a sodium silicate solution and then in a 1.5 SBF (simulated body fluid) solution. The crystallinity of the HA formed over the membranes was correlated to the use of the nucleation agent (the sodium silicate solution itself). Coated membranes were characterized by means of scanning electron microscopy – SEM, X-ray diffraction – XRD, and Fourier transform infrared spectroscopy – FTIR. The results indicate a homogeneous coating covering the entire surface of the membrane and the production of a semi-crystalline hydroxyapatite al ph layer similar to the miner . Introduction Interest in the use of chitosan for medical and pharmaceutical pplications has been growing, especially because of its remarkable roperties such as biocompatibility, which makes it suitable for everal medical applications [1,2]. The wide variety of shapes that chitosan can be delivered in, .g., films, membranes, fibers, gel, paste, tablets, microspheres, as ell as flakes, powders and solutions, enables it to be employed n several commercial, industrial, environmental and biomedical pplications [3]. Until a few years ago, chitosan was used mainly o remove sediments and metallic ions from water, as well as in he food industry. Currently, chitosan is used in the production of osmetics, medicines, food additives, and semi-permeable mem- ranes, and in the development of biomaterials for medical and ental applications. The development and use of several kinds of biomaterials in for issue reconstruction procedures are growing. Calcium phosphate eramics are used especially for this purpose due to their biocom- ∗ Corresponding author. E-mail address: edsonafilho@yahoo.com.br (E.d.A. Filho). 169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. oi:10.1016/j.apsusc.2010.11.104 ase of human bone. © 2010 Elsevier B.V. All rights reserved. patibility and osteocompatibility and their structural, physical and chemical similarity to the bone mineral matrix [4]. The use of these bioceramics does not induce any undesirable immunological or toxic reaction. There is no risk of transmission of infectious or contagious pathologies or of protein degradation because of their characteristics and their high purity, which is a result of the rigid control of the manufacturing process according to the strictest standards [5]. Hydroxyapatite is noteworthy because it is the main constituent of the mineral phase of calcified tissues. HA is a ceramic calcium phosphate, or bioceramic, whose structure and composition are similar to the mineral phase of bones and teeth [6]. Synthetic HA is also biocompatible and osteointegrable, which makes it one of the most important substitutes for the human bone in implants and prostheses. In technological applications, HA is used as a coating for metallic implants and periodontal membranes [7]. Relatively recent studies consider the use of chitosan as a bio- material because of its compatibility with live organisms and also due to economic reasons, since it derives from chitin, which is very abundant in nature. In view of the increasing use of chitosan as a biomaterial, the production of an organic mineral containing chi- tosan and hydroxyapatite should be studied, particularly due to the possible interactions between these constituents. The aim of dx.doi.org/10.1016/j.apsusc.2010.11.104 http://www.sciencedirect.com/science/journal/01694332 http://www.elsevier.com/locate/apsusc mailto:edsonafilho@yahoo.com.br dx.doi.org/10.1016/j.apsusc.2010.11.104 A.F. Fraga et al. / Applied Surface Science 257 (2011) 3888–3892 3889 Table 1 Ionic concentrations of the solutions used to coat apatites (mM) [9]. Na+ K+ Ca2+ Mg2+ HCO3 2− Cl− HPO4 2− SO4 2− SiO3 2− t c f 2 c 8 2 i s a w f s t t d i 2 b s p ( u S i S d S a a 2 s s e 2 a t a Blood plasma 142.0 5.0 2.5 1.5 SBF 142.0 5.0 2.5 1.5 1.5 SBF 213.0 7.5 3.8 2.3 Na2SiO3 2.0 – – – he present work is to study and produce chitosan membranes oated with hydroxyapatite by the modified biomimetic method or possible application as a biomaterial. . Materials and methods All the reagents used here were of analytical grade. Commercial hitosan (Sigma) was extracted from crab, and contained at least 5% of deacetylated chitin. .1. Production of chitosan membranes Chitosan membranes were produced from a solution contain- ng 2% of chitosan in acetic acid (weight/volume). This solution was tored for one week at 4 ◦C to allow for its complete solubilization ccording to the method proposed by Beppu et al. [8]. The solution as vacuum filtered to eliminate air bubbles and to prevent the ormation of macro-defects on the surface of the membranes after ynthesizing. The membranes were prepared by pouring the chi- osan solution into 2 cm × 3 cm TeflonTM molds with a controlled hickness of 3 mm. The molds containing the solution were then ried for 72 h at 40 ◦C, after which the membranes were immersed n 1 mol/L NaOH solution for 24 h at 25 ◦C. .2. Coating of chitosan membranes with hydroxyapatite In this work, we chose to use a modified version of the iomimetic method introduced by Abe et al. [5]. Rigo et al. [9] ubstituted the treatment with G glass from the original method roposed by Abe and coworkers by immersion in a sodium silicate SS) solution, which acts as a nucleation agent. [9,10] The membranes were coated with and without treatment in SS, sing the following routes: S1 – membranes were immersed in 1.5 BF solution for 3 days at 37 ◦C and pH = 7.0; S2 – membranes were mmersed in the SS solution for 3 days at 37 ◦C and then in 1.5 BF for 3 days at 37 ◦C and pH = 7.0, according to the procedure escribed by Rigo et al. [9]; S3 – membranes were immersed in the S solution for 3 days at 37 ◦C and then in 1.5 SBF for 7 days at 37 ◦C nd pH = 7.0. Table 1 lists the ionic concentrations of blood plasma nd all the solutions used in this process [10]. .3. Scanning electron microscopy A morphological analysis of the membranes was performed by canning electron microscopy (SEM), using a Philips TMP micro- cope. Organic specimens were coated with gold to improve their lectrical conductivity. .4. X-ray diffraction The presence of crystalline phases in the coated membranes was nalyzed by X-ray diffraction (XRD), using a Siemens D5005 diffrac- ometer with Cu (k�1) radiation and angular scanning between 4◦ nd 70◦. 27.0 103.0 1.0 0.5 – 4.2 148.0 1.0 0.5 – 6.3 223.0 1.5 0.75 – – – – – 3.6 2.5. Fourier transform infrared spectroscopy The ionic groups were characterized by vibrational infrared spectroscopy, using a Nicolet Magna 550 FTIR spectrophotometer with DRIFT CollectorTM diffuse reflectance. 3. Results and discussion The kinetic of formation of apatites via the biomimetic process using SBF solution follows the sequence shown in Eq. (1) [11]. (ACP) Ca10−xH2x(PO4)6(OH)2 → (OCP) Ca8H2(PO4)6 · 5H2O → (HA)Ca10(PO4)6(OH)2 (1) According to several authors, in the initial steps of apatite precipitation, low calcium content hydroxyapatite can become hydroxyapatite without transforming into octacalcium phosphate (OCP). Processing parameters such as pH (neutral or alkaline), tem- perature and solution concentration are very important for the crystallization of calcium phosphates until the HA phase is formed. Phenomena such as precipitation, solubilization, and slow and effective dehydration at a molecular level lead to the formation of a molecular arrangement that allows for the phase transformation of apatites [4]. Fig. 1 depicts the coatings produced in all the conditions employed in this study. The coating produced without the nucle- ation agent SS (condition S1) shows a dense homogeneous layer on the surface with an undefined morphology of ACP, which is one of the precursors of HA. The morphology of the HA coating pretreated with sodium silicate (condition S2) is dense and uniform, consist- ing of spherical particles of 1–5 �m, similar to those reported in the literature for HA coatings on metals [12,13]. The coating produced in condition S3 was dense. However, after 7 days of exposure to the 1.5 SBF solution, the coating showed surface cracks and some of its outer spherical particles were not bonded to inner particles. This indicates that the concentration at the surface of the coating became saturated when compared to the concentration of the 1.5 SBF solution [4]. After 4 days of exposure to the 1.5 SBF solution, the reaction at equilibrium changed from precipitation to dissolution of the HA layer. Fig. 2 shows the proposed coating mechanism of CS membranes with HA by means of the biomimetic method. Carbon in position 6 – C6 in CS has a free hydroxyl group that can help activation of the membrane surface by silanol groups of the SS solution. The silicate ions in the SS solution are adsorbed on the CS substrate (Fig. 2a); HA nucleation of the adsorbed silicate ions begins (Fig. 2b); HA nucleons grow because of the saturated 1.5 SBF solution, leading to the coating of the substrate (Fig. 2c). Hydroxyapatite Ca10(PO4)6(OH)2 has vibrational modes of phosphates and hydroxyls [14]. Table 2 shows the absorption fre- quencies for calcium phosphates. The spectrum in Fig. 3 shows HA absorption bands at 3570 cm−1 and at about 630 cm−1. The band at 3570 cm−1 is related to OH− stretching, which is attributed to the HA functional group. How- ever, this region shows broadening of the band, which increases with exposure time to the 1.5 SBF solution due to the water in the structure, a common occurrence in non-heat-treated coatings. 3890 A.F. Fraga et al. / Applied Surface Science 257 (2011) 3888–3892 Fig. 1. SEM characterization of chitosan/HA coated membranes. (S1) Membrane with no addition of SS and 3 days in 1.5 SBF; (S2) 3 days in SS and 3 days in 1.5 SBF; (S3) 3 days in SS and 3 days in 1.5 SBF. Fig. 2. Proposed coating mechanism of CS membranes with HA by means of biomimetic method. (a) The membrane is submitted to a treatment with sodium silicate solution; (b) membrane immersed in 1.5 SBF solution; (c) HA formation onto the surface. A.F. Fraga et al. / Applied Surface Science 257 (2011) 3888–3892 3891 Table 2 Characteristic calcium phosphates infrared absorption. Absorption band Wavenumber (cm−1) OH− 3572, 630 s C t p s d s w o P r 1 b e m S3 S2 OH OH OH P-OH P-OH P-OH P-O P-O P-O C-O C-O C-O C-C C-C C-C CO 2 CO 2 CO 2 C-H C-H C-H OH OH OH R e la ti v e I n te n s it y 4000 3500 3000 2500 2000 Wavenumber (cm-1) 1500 1000 500 S1 F d OH− (H2O) 3000–3700, 1600–1650 PO4 3− 474, 562, 580, 640 e 960–1200 P–OH 527, 870 e 910–1040 The bands at 2880 cm−1 (C–H stretching), 1660 cm−1 (C–C tretching) and 1390 cm−1 (C–O stretching) are attributed to the S structure. The band at 2330 cm−1 is attributed to the presence of CO2 in he measuring chamber of the device in which the specimen was laced, and is part of the equipment’s background noise. The bands at 1170 cm−1 and 1130 cm−1 are attributed to P–O tretching, while the bands close to 600 cm−1 are related to P–O eformation vibrations of the PO4 3− group [15]. The bands between 850 and 1000 cm−1 are attributed to P–OH tretching. A characteristic band of HA is visible at around 630 cm−1, hich represents the OH end-group with lower steric impediment f the structure [15]. The XRD measurements were taken using the PDF #39-194, DF #18-0303, and PDF #373-1731 standards for CS, ACP and HA, espectively [16]. A region of low crystallinity was visible between 5◦ and 25◦, corresponding to a diffraction halo of the CS mem- rane. The fact that the specimens showed low crystallinity is inter- sting since, according to Kanazawa [4], human bone consists of a ixture of calcium phosphates of low crystallinity. Discrete peaks ig. 4. XRD patterns for chitosan/HA membranes. (S1) Membrane with no addition of SS a ays in 1.5 SBF; (S2/bone) human bone and S2 condition. Fig. 3. FTIR spectra of chitosan/HA membranes. (S1) Membrane with no addition of SS and 3 days in 1.5 SBF; (S2) 3 days in SS and 3 days in 1.5 SBF; (S3) 3 days in SS and 3 days in 1.5 SBF. of the ACP phase (S1) close to 30◦ and 35◦ are shown in Fig. 4. The membrane pretreated in SS solution (S2) and exposed to the 1.5 SBF solution for 3 days shows peaks of low crystallinity related to the HA phase which are very similar to the ones Vercik [17] iden- tified in metallic substrates. This is a carbonated hydroxyapatite very similar to the biological one, which is characterized by its low crystallinity. nd 3 days in 1.5 SBF; (S2) 3 days in SS and 3 days in 1.5 SBF; (S3) 3 days in SS and 3 3 ace Sc d t r T o o w t o d m c b F 4 w o t a t f c [ [ [ [ [ [ 892 A.F. Fraga et al. / Applied Surf A tendency for higher crystallization was observed in the S3 con- ition. This behavior was expected because of the longer exposure o the 1.5 SBF solution (7 days). During that time, solubilization and eprecipitation of HA occurred, as indicated by the SEM analysis. his led to the slow formation of nuclei and the subsequent growth f more oriented crystals, favoring the formation of HA phase. Fig. 4 compares the biological HA of human bone and the results btained for the coating of S2 specimen. Back in 1926, De Jong [18] as the first to observe the similarity between X-ray spectra of he mineral phase of bone and HA. However, the mineral phase f bone does not have a definite composition because it varies uring the maturing and aging stages of hard tissues. Thus, the ain difference between synthetic HA and bone apatite lies in their rystallinity [19]. The similarity between diffractograms of human one and the one found experimentally (S2 condition) is shown in ig. 4. . Conclusions The modified biomimetic method proposed here enabled us to ork with the kinetics of the solution reactions, allowing for control f the degree of crystallinity of the HA layer. The use of sodium silicate as a nucleation agent in the produc- ion of hydroxyapatite coating affected the phase formation of the patites. After immersing the membranes in sodium silicate solu- ion, it was found that distinct calcium phosphate phases were ormed by varying the exposure time to 1.5 SBF solution. The longer the exposure time to the 1.5 SBF solution the more rystalline the coating and the lower its adhesion to the substrate. [ [ [ [ ience 257 (2011) 3888–3892 The conditions employed in this study led to the formation of a low crystallinity hydroxyapatite layer similar to human bone. Acknowledgments The authors would like to thank CNPq and FAPESP (Brazil) for their financial support of this work. References [1] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Eur. J. Pharm. Biopharm. 57 (2004) 19. [2] M.R. Finisie, A. Josue, V.T. Favere, M.C.M. Laranjeira, Anais da Academia Brasileira de Ciências 73 (2001) 525. [3] M.N.V.R. Kumar, React. Funct. Polym. 46 (2000) 1. [4] T. Kanazawa, Inorganic Phosphate materials, Elsevier, Tokyo, 1989, pp. 15–17. [5] Y. Abe, T. Kokubo, T. Yamamuro, J. Mater. Sci.: Mater. Med. 1 (1990) 536. [6] R.Z. Legeros, H.M. Myers, Monogr. Oral Sci. 15 (1991) 15. [7] E.Y. Kawachi, Quim. Nova 23 (2000) 518. [8] M.M. Beppu, E.J. Arruda, C.C. Santanam, Pol.: Ciên. e Tecn. (1999) 163. [9] E.C.S. Rigo, A.O. Boschi, M. Yoshimoto, Mater. Sci. Eng. C 24 (2004) 647. 10] J. Pierri, E.B. Roslindo, R. Tomasi, J. Non-Cryst. Solids 352 (2006) 5279. 11] H. Aoki, Science and Medical Applications of Hydroxyapatite, JAAS, Tokyo, 1991, p. 230. 12] H.M. Kim, F. Miyaji, T. Kokubo, C. Ohtsuki, T. Nakamura, J. Am. Ceram. Soc. 78 (1995) 2405. 13] T. Kokubo, Acta Mater. 46 (1998) 2519. 14] H. Zeng, W.R. Lacefeld, Biomaterials 21 (2000) 23. 15] J.C. Elliot, Structure and Chemistry of the Apatites and Other Calcium Orthophosphates, Elsevier, New York, 1984, p. 389. 16] Joint Committee for Powder Diffraction Studies – Diffraction Data Base. Inter- national for Diffraction Data, Newton Square, 2003. 1 CD-ROM. 17] L.C.O. Vercik, Doctoral Thesis, UNESP, 2004, p. 127. 18] W.F. De Jong, Recl. Trav. Chim. 45 (1926) 445. 19] A.F. Fraga, Master’s Dissertation, UFSCar, 2007, p. 120. Synthesis of chitosan/hydroxyapatite membranes coated with hydroxycarbonate apatite for guided tissue regeneration purposes Introduction Materials and methods Production of chitosan membranes Coating of chitosan membranes with hydroxyapatite Scanning electron microscopy X-ray diffraction Fourier transform infrared spectroscopy Results and discussion Conclusions Acknowledgments References