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Structural characterization of new chitosan-containing hybrid bioactive gels
Journal of Non-Crystalline Solids 351 (2005) 3037–3043 www.elsevier.com/locate/jnoncrysol
Structural characterization of new chitosan-containing hybrid bioactive gels Daniela Deriu a, Almerinda Di Venere a,b, Giampiero Mei Roberto Santucci a, Nicola Rosato a,b,* a
a,b
,
Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, 00133 Rome, Italy b INFM, National Institute for the Physics of Matter, Genova, Italy Received 25 February 2005; received in revised form 5 July 2005
Abstract The aim of the present study is the characterization of a new hybrid biomaterial obtained by combining a natural biopolymer, i.e chitosan, with an inorganic phase previously modified by the addition of calcium and phosphate ions. To this issue, we have prepared different chitosan-containing samples and tested both their solvent permeability and ion release capability. Data reveal the occurrence of a severe swelling process, whose kinetics is slowed down by the presence of increasing amounts of chitosan. Structural tests were performed by employing infrared spectroscopy and weight change measurements; further, the changes induced by chitosan on the biogel matrix were determinated by the use of confocal microscopy. Our results demonstrate that chitosan modifies the overall chemical structure of the inorganic phase, and reduces leaching effects without producing relevant interferences to the formation of the apatite layer on the bioactive gel surface. By defining more accurate limits to biopolymer incorporation into the bioactive gels, our data provide new insights on the potential offered by chitosan for incorporation into new biocompatible materials. 2005 Elsevier B.V. All rights reserved. PACS: 81.20.FW; 81.05.Kf; 61.43.Fs
1. Introduction In the last years, growing scientific effort has been made in developing new bioactive and biocompatible materials. These compounds can be synthesized through
Abbreviations: TMOS, Tetramethyl-orthosilicate; FTIR, Fouriertransform infrared spectroscopy; ICP, Inductive coupled plasma emission spectroscopy; Tris, Tris (hydroxymethyl)amino-methane; RubPy, Tris 2,2 0 bipyridyl dichlororuthenium(II) hexahydrate; HCA, Hydroxycarbonate apatite * Corresponding author. Address: Department of Experimental Medicine and Biochemical Sciences, University of Rome ÔTor VergataÕ, via Montpellier, 1-00133 Rome, Italy. Tel.: +39 06 72596460; fax: +39 06 72596468. E-mail address: [email protected] (N. Rosato). 0022-3093/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jnoncrysol.2005.07.013
a hybridization route, in which two different components, namely organic and inorganic, are combined. Sol–gel technology allows the incorporation of polymers of different nature in an inorganic silica bulk, thus producing organic–inorganic hybrid materials [1,2]. Depending on the strength of the interaction between the two phases, the hybrids can be divided in two different classes. The first one includes weak-bound components involving hydrogen or Van Der Waals bonds, whereas the second class of hybrids is characterized by strong covalent interactions [2]. Compared to the conventional melting process, the sol–gel methodology allows one to synthesize these hybrid materials at lower temperature, and provides a better control of their composition. Formation of the inorganic matrix (sol phase) is achieved by the complete, or partial, hydrolysis of a
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proper precursor, such as the tetramethyl-orthosilicate (TMOS) [3]. Depending on pH, the reaction leads to different bulk structures, which are less branched in acid condition, where the condensation rate is slower than that of hydrolysis (conversely the condensation rate is faster at alkaline pH). The reactions occurring during the sol–gel process are shown below [4]:
are discussed in the light of its potential employment in new biocompatible materials.
hydrolysis : nðSiORÞ4 þ 4nH2 O ! nðSiOHÞ4 þ 4nROH
Chitosan (molecular weight 70 000 Da), TMOS and ortho-phosphoric acid 85% were purchased from Fluka. Calcium nitrate and Tris 2,2 0 bipyridyl dichlororuthenium(II) hexahydrate (RubPy) were purchased from Sigma. All chemicals were reagent grade and used without further purification.
condensation :
nðSiOHÞ ! nSiO2 þ 2nH2 O
where R represents the alkyl group. Hybrid materials are instead obtained by incorporating also an organic phase, namely a natural or synthetic polymer [5,6]. These hybrids, which can be prepared as monoliths, disks or films, show chemical and physical features that generally differ from those of the precursor, showing an enhanced physiological tolerability. For this reason they find important application in biomedical, bioengineering, pharmacological and biosensor areas [7]. For instance, they have been recently employed in the construction of nano-composite polymers with particularly high performance [1]. A weak point of the sol–gel methodology is the fragility of the silica matrices. This problem can be partially overcome by incorporating organic polymers such as chitosan, polyethylene oxide, polyethylene glycol, polyvinyl alcohol, which provide stronger resiliency to the system [8]. The homogeneous dispersion of such soluble organic polymers during formation of the sol phase, strictly depends on the time at which the addition is made. Due to its peculiar features (non-toxicity, high biocompatibility and biodegradability) and to commercial availability, chitosan has been one of the most largely studied natural materials in the last years [9–11]. Several studies have pointed out its usefulness in the synthesis of hybrid membranes [12] and hybrid aerogels [13], that may find important application in the future development of pharmacological, biomedical and waste treatment products [14,15]. Recently, chitosan has been employed in calcium–phosphate enriched scaffolds glasses [16] and in hydrothermally hot pressed calcium silicate compacts [17], to achieve a significant reinforcement of these structures. In this paper we have analyzed the properties of hybrid materials containing different chitosan-to-silica ratios. The samples were synthesized by the sol–gel methodology, using an inorganic phase modified with calcium and phosphate ions, necessary to provide the glass surface with appropriate nucleation centers for mineralization. The interference of chitosan with this process has been studied using Fourier-transform infrared spectroscopy (FTIR) and inductive coupled plasma (ICP) emission spectroscopy. The restrictions to the amount of chitosan incorporated in new biomaterials
2. Experimental 2.1. Chemicals
2.2. Samples preparation A 0.1% (w/v) chitosan solution was prepared dissolving chitosan granules in a sodium acetate 0.1 M (pH = 5.0) solution, stirred vigorously on a heating plate for 5 h. The bioactive glasses were prepared according to the procedure described by Palumbo et al. [18]. In particular, the sol–gel mixture was obtained dissolving Ca(NO3)24H2O (1.68 g) in a 4.6 ml solution containing H3PO4 (0.164 g) and NaOH (0.0648 g). This mixture was added drop-wise to a TMOS solution (3.56 ml) contained in an ice-bathed flask, under magnetic stirring. Then, this solution was vigorously stirred for 50 min, at room temperature, and combined with three different amounts of chitosan. Tris–HCl buffer (pH = 7.2, 0.01 M) was then added to each sample in a constant proportion (40:60). For the three chitosan-containing samples the final chitosan-to-bioactive gel volumetric ratios were 2:3, 1:1, 2:1 and they have been respectively addressed as BK1, BK2 and BK3. As a control, with the same procedure a bioactive gel without chitosan was also prepared and addressed as sample B (Table 1). Each sample (2 ml in volume) was put in a Petri plate (35 · 10 mm) and placed for one week at room temperature in a dryer, provided with several water-containing beakers, to create a wet environment. After three more days in air the samples were ready for the analysis. Ion release was checked preparing several stocks of each dried sample (2 ml in volume) and soaking them with static immersion for different time intervals in separated 0.01 M Tris–HCl, pH = 7.2, solutions (6 ml in volume). The buffer was then removed to measure both the pH and the Ca2+ and PO3 4 content. The samples, washed and air-dried, were used for FTIR measurements. 2.3. Swelling measurements The swelling effect of the hybrids (due to water adsorption) has been characterized measuring their relative weight change. In these assays the samples were
D. Deriu et al. / Journal of Non-Crystalline Solids 351 (2005) 3037–3043 Table 1 Molar composition of the bioactive gel (sample B) Oxide
Initial (mmol)
Content (%)
P2O5 Na2O CaO N2O5 SiO2
0.83 0.57 7.11 7.11 23.6
2.1 1.5 18.1 18.1 60.2
soaked in a 6 ml 0.01 M Tris–HCl buffer (pH = 7.2) for different time intervals and then weighted. The degree of swelling was evaluated according to: wt (%) = (1 wtd/ wtw) · 100, where wtd and wtw represent the dry and wet weights, respectively. 2.4. Spectroscopic assays Infrared spectra of the hybrid materials were recorded in the range 4000–450 cm1 (100 scans), at 20 C, on a Philips PU 9800 spectrophotometer, using solid discs with the same thickness prepared with the KBr technique. The sample to KBr ratio (1:100) was the same for all measurements. Inductively coupled plasma spectroscopy (ICP) measurements were carried out on a Varian ICP, VISTA MPX model. Permeability studies were performed using a fluorescent probe, namely RubPy dissolved in a 0.5 ml Tris– HCl buffer. In particular, each sample was separately immersed in this solution and at determined time intervals the decrease in the fluorescence signal of the buffer measured on a photon-counting ISS-K2 spectrofluorometer (ISS, Champaign, USA), at 20 C (excitation wavelength = 454 nm, bandwidths = ±4 nm). 2.5. Confocal microscopy
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soaked for different time intervals in a solution containing a known concentration of RubPy. After adsorption of the probe, the residual amount of RubPy left in the soaking bath was determined spectroscopically, by measuring the fluorescence intensity. As shown in Fig. 1, after 30 min the amount of chitosan in the biogel significantly influences the residual fluorescence of the soaking bath; however, such effect appears considerably reduced at longer time intervals. Our results indicate that after 24 h the same amount of probe is absorbed in each sample, independently from the biogel composition. Thus, although the diffusion process is initially depending on the amount of chitosan, after 24 h the structural changes in the sample matrix allow a large solvent penetration effect. In a previous study, Ayers and Hunt [13] demonstrated that chitosan inhibits the gel shrinkage by reducing the superficial condensation reaction between adjacent silica particles. Samples with a high content of chitosan are therefore expected to reduce the compactness of the gel structure. As a consequence, hybrid biomaterials might adsorb a larger amount of water when in contact with buffer solutions and vary their specific volume. To shed deep light on this point, the four samples under investigation were immersed in a Tris–HCl solution for 6, 24, 48 and 54 h, and then weighed. Data obtained, shown in Fig. 2, indicate that a clear swelling effect takes place in the presence of chitosan, depending on the biopolymer concentration. In particular, as far as the bioactive gel-to-chitosan ratio is >1 (sample BK1) no relevant change is observed in the first 24 h (Fig. 2). On the other hand, for the samples containing higher chitosan concentration the process requires longer time to reach the equilibrium state (see data collected after 48 and 54 h, samples BK2 and BK3). An attempt to provide a reasonable interpretation for the data of Figs. 1 and 2
Surface images of the samples after immersion in Tris–HCl buffer for 48 h were acquired using a Nikon C1 confocal microscope excitated at 488 nm with an argon ion laser. The microphotographies were processed using a Nikon software EZ C1.
3. Results and discussion 3.1. Structural properties The synthesis of a hybrid bioactive gel might be in principle affected by the insertion of a large biopolymer such as chitosan. We have therefore carried out some preliminary tests to ascertain to which extent chitosan influences the overall structural properties of the bioactive gels. First of all, we have studied the permeability of the new material, observing the diffusion of a fluorescent probe across its matrix. In particular, the samples were
Fig. 1. Residual fluorescence of the soaking solution after RubPy adsorption by the bioactive gel (B) and by the three hybrid samples (BK1, BK2 and BK3) at different time intervals, namely 30 min (circles), 5 h (squares) and 24 h (triangles). The three lines represent the best linear fit.
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Fig. 2. Weight increase of four different samples, after: 6 h (dark gray bars), 24 h (dotted bars), 48 h (dashed bars), and 54 h (empty bars) from immersion in Tris–HCl soaking bath.
Fig. 4. FTIR spectra of sample B (line a) and of the chitosancontaining samples, namely BK1 (line b) and BK3 (line c). The spectrum of chitosan alone is also shown (line d).
are schematically represented in Fig. 3, where the enhanced diffusion at 24 h is attributed both to samples swelling and to the consequent change of the matrix network (third column). More structural details have been obtained by comparing the infrared spectrum of the bioactive gel with that of the hybrid material. As shown in Fig. 4, the fine structure of the bioactive gel spectrum remains unchanged upon chitosan incorporation, although the intensity of the main Si–O–Si (1100 cm1) and Si– OH (950 cm1) stretching bands decreases due to the minor silica content in the hybrid samples (Fig. 4, lines b and c). The additional absorption band observed around 1400 cm1 can be ascribed to the organic modi-
fication of the silica matrix and, in particular, to the formation of Si–C bonds [19]. On the other hand, larger changes occur in the spectrum profile of chitosan. The spectra of the hybrid samples in Fig. 4 (lines b and c) are mainly characterized by two effects: (i) the presence of a new absorption band observed around 1400 cm1, ascribable to the formation of Si–C bonds [19]; (ii) the absence of the typical amide II band (at 1570 cm1) of chitosan –NH bending mode, which is known to be right-shifted and considerably reduced in hybrid silica/ chitosan gels [10]. Interestingly the peak around 1640 cm1, characteristics of molecular water [20], does not change as far as the chitosan-to-silica ratio is kept below 1:1 (Fig. 4). This suggests that the amount of water entrapped in the original bioactive gel network is largely maintained within the BK1 sample. 3.2. Ion release
Fig. 3. Graphical representation of the structural changes likely induced in the bioactive gel by chitosan. The gray area indicates the increase (in percentage) of the sample size, due to the swelling process. Dots and lines depict RubPy and chitosan molecules, respectively.
A very important feature of bioactive glasses is their ability to form hydroxycarbonate apatite (HCA) on their surface, as they get in contact with physiological fluids. The HCA layer displays characteristics resembling those of the mineral component present in the bone matrix [Ca10(PO4)6(OH)2]. In vitro, formation of the HCA layer can be reproduced by soaking the bioactive glasses in an appropriate solution (for example in Tris–HCl buffer) which mimics the human plasma or other body fluids [21–25]. The mechanism of apatite formation is rather complex; it includes the release of different ions from the gel matrix and the successive re-adsorption of calcium and phosphate at the level of the gel surface [26]. Fig. 5 shows a leaching test of the bioactive gel alone (i.e. without chitosan), using three different soaking baths, namely a 0.9% (w/v) NaCl solution, de-ionized water and a Tris–HCl buffer. After 30 min the lowest amount of released ions was observed in the Tris–HCl buffer. Interestingly, in this last case a better preserva-
D. Deriu et al. / Journal of Non-Crystalline Solids 351 (2005) 3037–3043
Fig. 5. Ions release (calcium, empty bars; phosphate, dark bars; silica, dashed bars) from sample B in three different soaking solutions. The data on calcium and phosphate have been scaled by a factor of 300 and 200 respectively, for the sake of clarity.
tion of the inorganic matrix was also achieved, as demonstrated by the small amount of Si lost (Fig. 5). According to these data, the ion release process in the presence of chitosan was investigated in Tris–HCl buffer. The results obtained for calcium and phosphate at three different time intervals (0.5, 6 and 24 h), are shown in Fig. 6(a) and (b), respectively. As expected, in the first part of the process (0.5–6 h) the ions are progressively released in solution, reaching the maximum concentration in approximately 6 h; then, the trend is inverted. This behavior, which is in line with previous reports
Fig. 6. Calcium (panel a) and phosphate (panel b) release from the soaking solution, as a function of time (30 min, dark gray bars; 6 h, dotted bars; 24 h, dashed bars).
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on bioactive glasses [21], may be explained in terms of ions re-adsorption process, which generally leads to formation of an apatite layer on the samples surface. The percentage of re-adsorbed material within 24 h, i.e. I (%) = [I (6 h) I (24 h)]/I (6 h), has been evaluated for each sample, yielding 0.46 (B), 0.43 (BK1), 0.44 (BK2), 0.62 (BK3) in the case of calcium, and 0.38 (B), 0.40 (BK1), 0.38 (BK2), 0.60 (BK3) in the case of the phosphate ion. Thus, comparison of data collected in the presence and in absence of chitosan (Fig. 6(a) and (b)) demonstrates that the biopolymer prevents the loss of calcium and phosphate ions from the sample matrix and does not affect the re-adsorption rate (with the only exception of the BK3 sample). The confocal microscopy of samples B and BK1, after their immersion in Tris–HCl buffer for 48 h, is illustrated in Fig. 7. We observed that the chitosan-containing samples show crystalline aggregates on their surface, as shown in Fig. 8. Since the chemical composition
Fig. 7. Microphotographs of sample B (panel a) and BK1 (panel b) after immersion in Tris–HCl buffer solution for 48 h.
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tional structure (as shown by FTIR spectroscopy); (ii) diffusion across the hybrids structure is slowed down by the presence of chitosan in the short time range (i.e. 630 min); (iii) a good re-adsorbed-to-released ratio of calcium and phosphate ions is observed in the presence of chitosan, which suggests the likely formation of a HCA layer at the hybrid surface; (iv) the samples investigated display a time-dependent swelling effect in Tris–HCl buffer; the equilibrium state is in fact reached within 24 h only in the case of the BK1 sample (that containing the low amount of chitosan). All together, these results provide new insights on the advantages (and limits) of chitosan-containing hybrid materials produced via sol–gel methodology.
Acknowledgments Authors wish to thank Dr R. Ferrante and Dr M. Pietroletti of the Chemical Department of the University of Rome ÔLa SapienzaÕ for assistance in the ICP and FTIR measurements, and Dr M. Ranalli of the Department of Experimental Medicine and Biochemical Sciences, University of Rome ÔTor VergataÕ, for assistance in confocal microscopy image acquisition. This study was partially supported by grants from Italian MIUR (PRIN 2004 055484).
References
Fig. 8. Crystalline aggregates on BK1 (panel a) and BK2 (panel b) surface after immersion in Tris–HCl buffer solution for 48 h.
of such crystals has not been determined, we cannot state that they are unequivocally composed by hydroxycarbonate apatite; however, we observed that the amount of crystals increases with the chitosan concentration (see panels a and b of Fig. 8). Finally, it is of interest the observation that the samples containing high amount of chitosan show a reduced growth of fungi on their surface, in full accord with previous reports [27,28].
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
4. Conclusions In conclusion, this study shows that novel chitosancontaining hybrids can be easily achieved by using an inorganic phase modified by the addition of calcium and phosphate ions. Our results suggest that: (i) chitosan slightly interferes with the inorganic bioactive gel network, which practically retains its overall fine vibra-
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D. Deriu et al. / Journal of Non-Crystalline Solids 351 (2005) 3037–3043 [21] M. Vallet-Regi, A. Ramila, Chem. Mater. 12 (2000) 961. [22] L.L. Hence, J. Biomed. Mater. Res. 41 (1998) 511. [23] D. Arcos, D.C. Greenspan, M. Vallet-Regi, J. Biomed. Mater. 65A (2003) 344. [24] R. Jones Julian, L.L. Hence, Curr. Opin. Solid State Mater. Sci. 7 (2003) 301.
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[25] E. Kontonasaki, T. Zorba, L. Papadopoulou, E. Pavlidou, X. Chatzistavrou, P. Koidis, Cryst. Res. Technol. 37 (2002) 1165. [26] Z. Kai, H. Yan, D.C. Bell, A. Stein, L.F. Francis, J. Biomed. Mater. Res. 66 (2003) 860. [27] S. Roller, N. Covill, Int. J. Food Microbiol. 47 (1999) 67. [28] L.-Y. Zheng, J.-F. Zhu, Carbohyd. Polym. 54 (2003) 527.
Structural characterization of new chitosan-containing hybrid bioactive gels Daniela Deriu a, Almerinda Di Venere a,b, Giampiero Mei Roberto Santucci a, Nicola Rosato a,b,* a
a,b
,
Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, 00133 Rome, Italy b INFM, National Institute for the Physics of Matter, Genova, Italy Received 25 February 2005; received in revised form 5 July 2005
Abstract The aim of the present study is the characterization of a new hybrid biomaterial obtained by combining a natural biopolymer, i.e chitosan, with an inorganic phase previously modified by the addition of calcium and phosphate ions. To this issue, we have prepared different chitosan-containing samples and tested both their solvent permeability and ion release capability. Data reveal the occurrence of a severe swelling process, whose kinetics is slowed down by the presence of increasing amounts of chitosan. Structural tests were performed by employing infrared spectroscopy and weight change measurements; further, the changes induced by chitosan on the biogel matrix were determinated by the use of confocal microscopy. Our results demonstrate that chitosan modifies the overall chemical structure of the inorganic phase, and reduces leaching effects without producing relevant interferences to the formation of the apatite layer on the bioactive gel surface. By defining more accurate limits to biopolymer incorporation into the bioactive gels, our data provide new insights on the potential offered by chitosan for incorporation into new biocompatible materials. 2005 Elsevier B.V. All rights reserved. PACS: 81.20.FW; 81.05.Kf; 61.43.Fs
1. Introduction In the last years, growing scientific effort has been made in developing new bioactive and biocompatible materials. These compounds can be synthesized through
Abbreviations: TMOS, Tetramethyl-orthosilicate; FTIR, Fouriertransform infrared spectroscopy; ICP, Inductive coupled plasma emission spectroscopy; Tris, Tris (hydroxymethyl)amino-methane; RubPy, Tris 2,2 0 bipyridyl dichlororuthenium(II) hexahydrate; HCA, Hydroxycarbonate apatite * Corresponding author. Address: Department of Experimental Medicine and Biochemical Sciences, University of Rome ÔTor VergataÕ, via Montpellier, 1-00133 Rome, Italy. Tel.: +39 06 72596460; fax: +39 06 72596468. E-mail address: [email protected] (N. Rosato). 0022-3093/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jnoncrysol.2005.07.013
a hybridization route, in which two different components, namely organic and inorganic, are combined. Sol–gel technology allows the incorporation of polymers of different nature in an inorganic silica bulk, thus producing organic–inorganic hybrid materials [1,2]. Depending on the strength of the interaction between the two phases, the hybrids can be divided in two different classes. The first one includes weak-bound components involving hydrogen or Van Der Waals bonds, whereas the second class of hybrids is characterized by strong covalent interactions [2]. Compared to the conventional melting process, the sol–gel methodology allows one to synthesize these hybrid materials at lower temperature, and provides a better control of their composition. Formation of the inorganic matrix (sol phase) is achieved by the complete, or partial, hydrolysis of a
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D. Deriu et al. / Journal of Non-Crystalline Solids 351 (2005) 3037–3043
proper precursor, such as the tetramethyl-orthosilicate (TMOS) [3]. Depending on pH, the reaction leads to different bulk structures, which are less branched in acid condition, where the condensation rate is slower than that of hydrolysis (conversely the condensation rate is faster at alkaline pH). The reactions occurring during the sol–gel process are shown below [4]:
are discussed in the light of its potential employment in new biocompatible materials.
hydrolysis : nðSiORÞ4 þ 4nH2 O ! nðSiOHÞ4 þ 4nROH
Chitosan (molecular weight 70 000 Da), TMOS and ortho-phosphoric acid 85% were purchased from Fluka. Calcium nitrate and Tris 2,2 0 bipyridyl dichlororuthenium(II) hexahydrate (RubPy) were purchased from Sigma. All chemicals were reagent grade and used without further purification.
condensation :
nðSiOHÞ ! nSiO2 þ 2nH2 O
where R represents the alkyl group. Hybrid materials are instead obtained by incorporating also an organic phase, namely a natural or synthetic polymer [5,6]. These hybrids, which can be prepared as monoliths, disks or films, show chemical and physical features that generally differ from those of the precursor, showing an enhanced physiological tolerability. For this reason they find important application in biomedical, bioengineering, pharmacological and biosensor areas [7]. For instance, they have been recently employed in the construction of nano-composite polymers with particularly high performance [1]. A weak point of the sol–gel methodology is the fragility of the silica matrices. This problem can be partially overcome by incorporating organic polymers such as chitosan, polyethylene oxide, polyethylene glycol, polyvinyl alcohol, which provide stronger resiliency to the system [8]. The homogeneous dispersion of such soluble organic polymers during formation of the sol phase, strictly depends on the time at which the addition is made. Due to its peculiar features (non-toxicity, high biocompatibility and biodegradability) and to commercial availability, chitosan has been one of the most largely studied natural materials in the last years [9–11]. Several studies have pointed out its usefulness in the synthesis of hybrid membranes [12] and hybrid aerogels [13], that may find important application in the future development of pharmacological, biomedical and waste treatment products [14,15]. Recently, chitosan has been employed in calcium–phosphate enriched scaffolds glasses [16] and in hydrothermally hot pressed calcium silicate compacts [17], to achieve a significant reinforcement of these structures. In this paper we have analyzed the properties of hybrid materials containing different chitosan-to-silica ratios. The samples were synthesized by the sol–gel methodology, using an inorganic phase modified with calcium and phosphate ions, necessary to provide the glass surface with appropriate nucleation centers for mineralization. The interference of chitosan with this process has been studied using Fourier-transform infrared spectroscopy (FTIR) and inductive coupled plasma (ICP) emission spectroscopy. The restrictions to the amount of chitosan incorporated in new biomaterials
2. Experimental 2.1. Chemicals
2.2. Samples preparation A 0.1% (w/v) chitosan solution was prepared dissolving chitosan granules in a sodium acetate 0.1 M (pH = 5.0) solution, stirred vigorously on a heating plate for 5 h. The bioactive glasses were prepared according to the procedure described by Palumbo et al. [18]. In particular, the sol–gel mixture was obtained dissolving Ca(NO3)24H2O (1.68 g) in a 4.6 ml solution containing H3PO4 (0.164 g) and NaOH (0.0648 g). This mixture was added drop-wise to a TMOS solution (3.56 ml) contained in an ice-bathed flask, under magnetic stirring. Then, this solution was vigorously stirred for 50 min, at room temperature, and combined with three different amounts of chitosan. Tris–HCl buffer (pH = 7.2, 0.01 M) was then added to each sample in a constant proportion (40:60). For the three chitosan-containing samples the final chitosan-to-bioactive gel volumetric ratios were 2:3, 1:1, 2:1 and they have been respectively addressed as BK1, BK2 and BK3. As a control, with the same procedure a bioactive gel without chitosan was also prepared and addressed as sample B (Table 1). Each sample (2 ml in volume) was put in a Petri plate (35 · 10 mm) and placed for one week at room temperature in a dryer, provided with several water-containing beakers, to create a wet environment. After three more days in air the samples were ready for the analysis. Ion release was checked preparing several stocks of each dried sample (2 ml in volume) and soaking them with static immersion for different time intervals in separated 0.01 M Tris–HCl, pH = 7.2, solutions (6 ml in volume). The buffer was then removed to measure both the pH and the Ca2+ and PO3 4 content. The samples, washed and air-dried, were used for FTIR measurements. 2.3. Swelling measurements The swelling effect of the hybrids (due to water adsorption) has been characterized measuring their relative weight change. In these assays the samples were
D. Deriu et al. / Journal of Non-Crystalline Solids 351 (2005) 3037–3043 Table 1 Molar composition of the bioactive gel (sample B) Oxide
Initial (mmol)
Content (%)
P2O5 Na2O CaO N2O5 SiO2
0.83 0.57 7.11 7.11 23.6
2.1 1.5 18.1 18.1 60.2
soaked in a 6 ml 0.01 M Tris–HCl buffer (pH = 7.2) for different time intervals and then weighted. The degree of swelling was evaluated according to: wt (%) = (1 wtd/ wtw) · 100, where wtd and wtw represent the dry and wet weights, respectively. 2.4. Spectroscopic assays Infrared spectra of the hybrid materials were recorded in the range 4000–450 cm1 (100 scans), at 20 C, on a Philips PU 9800 spectrophotometer, using solid discs with the same thickness prepared with the KBr technique. The sample to KBr ratio (1:100) was the same for all measurements. Inductively coupled plasma spectroscopy (ICP) measurements were carried out on a Varian ICP, VISTA MPX model. Permeability studies were performed using a fluorescent probe, namely RubPy dissolved in a 0.5 ml Tris– HCl buffer. In particular, each sample was separately immersed in this solution and at determined time intervals the decrease in the fluorescence signal of the buffer measured on a photon-counting ISS-K2 spectrofluorometer (ISS, Champaign, USA), at 20 C (excitation wavelength = 454 nm, bandwidths = ±4 nm). 2.5. Confocal microscopy
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soaked for different time intervals in a solution containing a known concentration of RubPy. After adsorption of the probe, the residual amount of RubPy left in the soaking bath was determined spectroscopically, by measuring the fluorescence intensity. As shown in Fig. 1, after 30 min the amount of chitosan in the biogel significantly influences the residual fluorescence of the soaking bath; however, such effect appears considerably reduced at longer time intervals. Our results indicate that after 24 h the same amount of probe is absorbed in each sample, independently from the biogel composition. Thus, although the diffusion process is initially depending on the amount of chitosan, after 24 h the structural changes in the sample matrix allow a large solvent penetration effect. In a previous study, Ayers and Hunt [13] demonstrated that chitosan inhibits the gel shrinkage by reducing the superficial condensation reaction between adjacent silica particles. Samples with a high content of chitosan are therefore expected to reduce the compactness of the gel structure. As a consequence, hybrid biomaterials might adsorb a larger amount of water when in contact with buffer solutions and vary their specific volume. To shed deep light on this point, the four samples under investigation were immersed in a Tris–HCl solution for 6, 24, 48 and 54 h, and then weighed. Data obtained, shown in Fig. 2, indicate that a clear swelling effect takes place in the presence of chitosan, depending on the biopolymer concentration. In particular, as far as the bioactive gel-to-chitosan ratio is >1 (sample BK1) no relevant change is observed in the first 24 h (Fig. 2). On the other hand, for the samples containing higher chitosan concentration the process requires longer time to reach the equilibrium state (see data collected after 48 and 54 h, samples BK2 and BK3). An attempt to provide a reasonable interpretation for the data of Figs. 1 and 2
Surface images of the samples after immersion in Tris–HCl buffer for 48 h were acquired using a Nikon C1 confocal microscope excitated at 488 nm with an argon ion laser. The microphotographies were processed using a Nikon software EZ C1.
3. Results and discussion 3.1. Structural properties The synthesis of a hybrid bioactive gel might be in principle affected by the insertion of a large biopolymer such as chitosan. We have therefore carried out some preliminary tests to ascertain to which extent chitosan influences the overall structural properties of the bioactive gels. First of all, we have studied the permeability of the new material, observing the diffusion of a fluorescent probe across its matrix. In particular, the samples were
Fig. 1. Residual fluorescence of the soaking solution after RubPy adsorption by the bioactive gel (B) and by the three hybrid samples (BK1, BK2 and BK3) at different time intervals, namely 30 min (circles), 5 h (squares) and 24 h (triangles). The three lines represent the best linear fit.
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Fig. 2. Weight increase of four different samples, after: 6 h (dark gray bars), 24 h (dotted bars), 48 h (dashed bars), and 54 h (empty bars) from immersion in Tris–HCl soaking bath.
Fig. 4. FTIR spectra of sample B (line a) and of the chitosancontaining samples, namely BK1 (line b) and BK3 (line c). The spectrum of chitosan alone is also shown (line d).
are schematically represented in Fig. 3, where the enhanced diffusion at 24 h is attributed both to samples swelling and to the consequent change of the matrix network (third column). More structural details have been obtained by comparing the infrared spectrum of the bioactive gel with that of the hybrid material. As shown in Fig. 4, the fine structure of the bioactive gel spectrum remains unchanged upon chitosan incorporation, although the intensity of the main Si–O–Si (1100 cm1) and Si– OH (950 cm1) stretching bands decreases due to the minor silica content in the hybrid samples (Fig. 4, lines b and c). The additional absorption band observed around 1400 cm1 can be ascribed to the organic modi-
fication of the silica matrix and, in particular, to the formation of Si–C bonds [19]. On the other hand, larger changes occur in the spectrum profile of chitosan. The spectra of the hybrid samples in Fig. 4 (lines b and c) are mainly characterized by two effects: (i) the presence of a new absorption band observed around 1400 cm1, ascribable to the formation of Si–C bonds [19]; (ii) the absence of the typical amide II band (at 1570 cm1) of chitosan –NH bending mode, which is known to be right-shifted and considerably reduced in hybrid silica/ chitosan gels [10]. Interestingly the peak around 1640 cm1, characteristics of molecular water [20], does not change as far as the chitosan-to-silica ratio is kept below 1:1 (Fig. 4). This suggests that the amount of water entrapped in the original bioactive gel network is largely maintained within the BK1 sample. 3.2. Ion release
Fig. 3. Graphical representation of the structural changes likely induced in the bioactive gel by chitosan. The gray area indicates the increase (in percentage) of the sample size, due to the swelling process. Dots and lines depict RubPy and chitosan molecules, respectively.
A very important feature of bioactive glasses is their ability to form hydroxycarbonate apatite (HCA) on their surface, as they get in contact with physiological fluids. The HCA layer displays characteristics resembling those of the mineral component present in the bone matrix [Ca10(PO4)6(OH)2]. In vitro, formation of the HCA layer can be reproduced by soaking the bioactive glasses in an appropriate solution (for example in Tris–HCl buffer) which mimics the human plasma or other body fluids [21–25]. The mechanism of apatite formation is rather complex; it includes the release of different ions from the gel matrix and the successive re-adsorption of calcium and phosphate at the level of the gel surface [26]. Fig. 5 shows a leaching test of the bioactive gel alone (i.e. without chitosan), using three different soaking baths, namely a 0.9% (w/v) NaCl solution, de-ionized water and a Tris–HCl buffer. After 30 min the lowest amount of released ions was observed in the Tris–HCl buffer. Interestingly, in this last case a better preserva-
D. Deriu et al. / Journal of Non-Crystalline Solids 351 (2005) 3037–3043
Fig. 5. Ions release (calcium, empty bars; phosphate, dark bars; silica, dashed bars) from sample B in three different soaking solutions. The data on calcium and phosphate have been scaled by a factor of 300 and 200 respectively, for the sake of clarity.
tion of the inorganic matrix was also achieved, as demonstrated by the small amount of Si lost (Fig. 5). According to these data, the ion release process in the presence of chitosan was investigated in Tris–HCl buffer. The results obtained for calcium and phosphate at three different time intervals (0.5, 6 and 24 h), are shown in Fig. 6(a) and (b), respectively. As expected, in the first part of the process (0.5–6 h) the ions are progressively released in solution, reaching the maximum concentration in approximately 6 h; then, the trend is inverted. This behavior, which is in line with previous reports
Fig. 6. Calcium (panel a) and phosphate (panel b) release from the soaking solution, as a function of time (30 min, dark gray bars; 6 h, dotted bars; 24 h, dashed bars).
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on bioactive glasses [21], may be explained in terms of ions re-adsorption process, which generally leads to formation of an apatite layer on the samples surface. The percentage of re-adsorbed material within 24 h, i.e. I (%) = [I (6 h) I (24 h)]/I (6 h), has been evaluated for each sample, yielding 0.46 (B), 0.43 (BK1), 0.44 (BK2), 0.62 (BK3) in the case of calcium, and 0.38 (B), 0.40 (BK1), 0.38 (BK2), 0.60 (BK3) in the case of the phosphate ion. Thus, comparison of data collected in the presence and in absence of chitosan (Fig. 6(a) and (b)) demonstrates that the biopolymer prevents the loss of calcium and phosphate ions from the sample matrix and does not affect the re-adsorption rate (with the only exception of the BK3 sample). The confocal microscopy of samples B and BK1, after their immersion in Tris–HCl buffer for 48 h, is illustrated in Fig. 7. We observed that the chitosan-containing samples show crystalline aggregates on their surface, as shown in Fig. 8. Since the chemical composition
Fig. 7. Microphotographs of sample B (panel a) and BK1 (panel b) after immersion in Tris–HCl buffer solution for 48 h.
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tional structure (as shown by FTIR spectroscopy); (ii) diffusion across the hybrids structure is slowed down by the presence of chitosan in the short time range (i.e. 630 min); (iii) a good re-adsorbed-to-released ratio of calcium and phosphate ions is observed in the presence of chitosan, which suggests the likely formation of a HCA layer at the hybrid surface; (iv) the samples investigated display a time-dependent swelling effect in Tris–HCl buffer; the equilibrium state is in fact reached within 24 h only in the case of the BK1 sample (that containing the low amount of chitosan). All together, these results provide new insights on the advantages (and limits) of chitosan-containing hybrid materials produced via sol–gel methodology.
Acknowledgments Authors wish to thank Dr R. Ferrante and Dr M. Pietroletti of the Chemical Department of the University of Rome ÔLa SapienzaÕ for assistance in the ICP and FTIR measurements, and Dr M. Ranalli of the Department of Experimental Medicine and Biochemical Sciences, University of Rome ÔTor VergataÕ, for assistance in confocal microscopy image acquisition. This study was partially supported by grants from Italian MIUR (PRIN 2004 055484).
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Fig. 8. Crystalline aggregates on BK1 (panel a) and BK2 (panel b) surface after immersion in Tris–HCl buffer solution for 48 h.
of such crystals has not been determined, we cannot state that they are unequivocally composed by hydroxycarbonate apatite; however, we observed that the amount of crystals increases with the chitosan concentration (see panels a and b of Fig. 8). Finally, it is of interest the observation that the samples containing high amount of chitosan show a reduced growth of fungi on their surface, in full accord with previous reports [27,28].
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4. Conclusions In conclusion, this study shows that novel chitosancontaining hybrids can be easily achieved by using an inorganic phase modified by the addition of calcium and phosphate ions. Our results suggest that: (i) chitosan slightly interferes with the inorganic bioactive gel network, which practically retains its overall fine vibra-
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