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Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method
Biomaterials 20 (1999) 879—884
Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method H.K. Varma* , Y. Yokogawa, F.F. Espinosa, Y. Kawamoto, K. Nishizawa, F. Nagata, T. Kameyama Bioceramic Laboratory, National Industrial Research Institute of Nagoya (NIRIN), Hirate-Cho, Kita-ku, Nagoya 462, Japan Received 26 May 1998; accepted 8 December 1998
Abstract A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energydispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H PO with the P content being 0.1—0.2 wt%. Phosphorylated films soaked in saturated Ca(OH) solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5;SBF for more than 20 days. Phosphorylated films not treated with Ca(OH) did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time. 1999 Elsevier Science Ltd. All rights reserved Keywords: Calcium phosphate; Biomimetic; Chitosan; Coating
1. Introduction Calcium-phosphate-based bioceramics, in the form of powder, granules and coatings are currently used for a number of dental and skeletal prosthetic applications by virtue of their excellent biocompatibility and osteointegration properties [1]. Recently, calcium phosphate materials are being grown biomimetically over different surface-modified substrates in order to gain insight into how to develop biomaterials with tailored surface properties that can initiate calcium phosphate deposition when implanted at bony sites [2—4]. This process has many advantages over conventional coating techniques and can be carried out at ambient reaction conditions.
* Corresponding author. Fax: #471 341 814. Permanent address: Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum 695 012, India.
The nucleation or precipitation of hydroxy apatite over different material surfaces can be achieved by either (a) raising the ionic activity product of calcium phosphate in the solution, thereby stimulating precipitation and creation of apatite nucleation sites [5] or by a (b) surface functionalization method, used to create favourable local conditions that lead to the nucleation and growth of calcium phosphate [6—8]. Deposition of calcium phosphate material over various biopolymeric films has been achieved by biomimetic approach by many researchers with a view to find suitable biomedical applications. These techniques are mainly based on functionalization and subsequent immersion of the substrate material in simulated body fluid solution (SBF). Phosphate group as the functionalization radical was found to be very effective for the deposition of HAP over cotton, chitin, etc. [6, 7]. Prior to immersion in SBF, the phosphorylated substrate is subjected to soaking in saturated Ca(OH) solution. This treatment creates a number of calcium phosphate precursor sites over the surface which will eventually create favourable condition for the nucleation and growth of HAP.
0142-9612/99/$ — see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 9 8 ) 0 0 2 4 3 - 9
880
H.K. Varma et al. / Biomaterials 20 (1999) 879—884
This work describes a method for depositing calcium phosphate mineral over surface-modified chitosan film. Chitosan is a polysaccharide (polymer of 2-amino 2-Dglucose) and is derived by deacetylation of chitin, which is the principal constituent in shells of crabs, lobsters and other crustaceans. It is currently being used in a number of medical applications such as drug encapsulation, fat absorber, and wound dressing materials to name a few. It is non-toxic and biocompatible. It was reported that chitosan phosphate is a very good cation absorber. Ions such as calcium, copper, cadmium, uranium, etc., from solutions were absorbed by phosphorylated chitosan and chitin [9], chitosan phosphate being a better absorbent than chitin phosphate. In the present work, phosphorylated chitosan films soaked in lime were immersed in 1.5;simulated body fluid (SBF) and examined for the calcium phosphate growth over their surfaces at different points in time. 2. Experiments 2.1. Materials All chemicals were supplied by Wako Pure Chemicals Co. and were used without further purification. The growth medium (1.5;SBF) was prepared by procedures reported elsewhere [4], and contained 15 ml of each of the following: 2.74 mol l\ NaCl, 0.06 mol l\ KCl, 0.05 mol l\CaCl , 0.03 mol l\ MgCl , 0.0895 mol l\ NaHCO , 0.02 mol l\ K HPO and 0.01 mol l\ Na SO . These were added to a 200 ml volumetric flask along with 25 ml each of 0.4 mol l\ Tris hydroxy methyl methane amine and 0.36 mol l\ of HCl. The pH of the solution was adjusted to 7.3 by adding a few drops of HCl with the remainder of the volume being distilled water. 2.2. Preparation of chitosan film Chitosan film was made by first dissolving 2 g of commercial chitosan powder in 100 ml of 2% acetic acid solution. The viscous liquid was then poured into glass petri dishes and kept in a vacuum oven at 700 mmHg and 35°C for 6 h to clear all the entrapped gas bubbles. The dishes were then transferred into an air oven and dried at 60°C for 24 h. The chitosan film was easily pealed off when saturated NaOH solution was introduced into the dishes. The wet films were then thoroughly rinsed with distilled water. Thickness of the films ranged between 0.2 and 0.3 mm.
a round-bottomed flask fitted with a condenser, thermometer and nitrogen gas inlet tube. The bottle was then heated to 120°C with the reactants refluxed for 1 h under gentle stirring using a magnetic stirrer. Some foam formation occurred inside the flask during the course of the reaction. After cooling the contents, the films were removed and thoroughly rinsed with distilled water. 2.4. Soaking in saturated Ca(OH)2 solution Some phosphorylated films were suspended in freshly prepared saturated Ca(OH) solution for a period of 8 days. The solution was replaced with fresh Ca(OH) after 3 days. After treatment, the films were thoroughly washed with distilled water. 2.5. Soaking in 1.5;SBF solution Lime soaked, phosphorylated chitosan films were vertically suspended in plastic jars with cotton threads and 50 ml of 1.5;SBF solution was added. The films were freely suspended and did not stick to each other. The jars were then covered with an airtight cap and put into an air oven kept at 36.5°C. The SBF solution was replaced each day. Samples were retrieved after 3, 7, 21 and 30 days of soaking. The retrieved samples were thoroughly rinsed with distilled water and dried at 65°C before performing various characterization studies.
3. Characterization Scanning electron microscopy and EDAX analysis were carried out using a Hitachi S530 scanning electron microscope and a Horiba EMAX 2200 X-ray micro analyzer. Micro-FTIR measurements were done in a Jasco Micro FTIR Jansen Fourier transform infrared spectrometer. Film samples were encased in 1 mm transparent KBr pellets in order to view under the FTIR microscope. The P content of the phosphorylated film and Ca and P content of the Ca(OH) -treated and SBF-treated films were determined using ICP analysis (Nippon Jarrell-Ash ICAP-1000S ICP-AES). Thin film X-ray diffraction studies were recorded using a MXP Material Analysis and Characterization XRD spectrometer using Cu K radiation. ? 4. Results
2.3. Phosphorylation of chitosan films 4.1. Phosphorylated chitosan film The phosphorylation reaction was carried out by a similar procedure reported earlier [9]. Approximately 1 g of chitosan film, 3 g of 98% H PO , 15 g of urea and 30 ml of dimethyl formamide were mixed together in
Untreated and phosphorylated chitosan films were transparent. Figure 1a shows an SEM photograph of the chitosan film. At high magnification, phosphorylated
H.K. Varma et al. / Biomaterials 20 (1999) 879—884
881
Fig. 1. Scanning electron micrograph of chitosan film (a) before and (b) after phosphorylation.
Fig. 3. Scanning electron micrograph of chitosan film after phosphorylation and Ca(OH) soaking for 8 days.
Fig. 2. Micro-FTIR spectrum of (a) chitosan film (b) chitosan film after phosphorylation and (c) after phosphorylation and Ca(OH) soaking for 8 days.
films showed small blisters and bubbles on their film surface (Fig. 1b). EDAX analysis of the above film revealed that the surface is rich in P. As per the ICP analysis of the film, P content is 0.1—0.2 wt%. This value is lower than the one reported for chitosan powder subjected to similar phosphorylation experiments and in that case, the P content was about 6 wt% [9]. Figure 2 shows the micro-FTIR spectrum of the chitosan film before and after phosphorylation. Peaks at 1650 and 1544 cm\ are from the amide bond and the 1000 cm\ is due to the C"O stretching due to the undeacetylated groups of the chitosan (Fig. 2a). The C—O stretch at 899 cm\ is also present. The 1510 and 1370 cm\ are
due to the primary amino group in chitosan. In the pattern for phosphorylated film (Fig. 2b), the PO vibra tion peaks are predominant compared to the existing chitosan peaks. Absorption peaks at 1150 and 1022 cm\ are due to the P—O stretch and 620 cm\ is due to the P—O bend. 4.2. Ca(OH)2-treated phosphorylated chitosan film When phosphorylated chitosan film was soaked in saturated CaO solution for 8 days, a coating of fine particles formed over its surface (Fig. 3), which were determined by EDAX to be calcium phosphate. As per the ICP data obtained for P and Ca, Ca/P ratio was 1.34. The micro-FTIR spectrum (Fig. 2c) showed a broad pattern of poorly crystalline calcium phosphate with 1150 and 620 cm\ peaks ascribed to P—O stretch and bend and are present along with the other chitosan peaks.
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H.K. Varma et al. / Biomaterials 20 (1999) 879—884
4.3. SBF treatment The Ca(OH) -treated phosphorylated chitosan films were immersed in 1.5;SBF solution for different periods of time. Figure 4 shows SEM pictures of the samples retrieved after 3, 7 and 21 days. After 3 days, a single layer of particles of calcium phosphate ((200 nm in size) started depositing over the surface. After 7 days, the surface is almost covered by calcium phosphate particles accompanied by secondary nucleation over the initial layer. This results in a porous aggregate of particles. Within 21 days, the entire surface was covered with
a porous calcium phosphate coating (Fig. 4c). EDAX analysis showed that these particles are calcium phosphate with Ca/P ratio less than 1.67, less than the Ca/P value of pure calcium hydroxy apatite. As the immersion time increases, the Ca/P ratio also increases (Fig. 5). The micro-FTIR spectrum of the phosphate-rich area showed that the coating is poorly crystallized hydroxy apatite. The major peaks at 1100 and 600 cm\ correspond to the PO group. The apatite is a carbonate substituted one as the carbonate peak at 1400 cm\ is quite predominant. Thin-film XRD patterns of the films immersed in SBF for 7 and 21 days are given in Fig. 6. The broad
Fig. 4. Low and high magnification scanning electron micrographs of phosphorylated and Ca(OH) treated chitosan films after immersion in 1.5;SBF solution at 36.5°C for (a and b) 3 days (c and d) 7 days and (e and f ) 21 days.
H.K. Varma et al. / Biomaterials 20 (1999) 879—884
Fig. 5. Plot of Ca/P ratio of the coating produced vs. time of immersion in 1.5;SBF solution for the phosphorylated and Ca(OH) treated chitosan films.
peak of 2h around 32—34° is ascribed to the diffraction of 211, 300 and 202 planes of HAP crystals. Another broad peak around 20° is due to the substrate chitosan film. A more resolved spectrum is obtained for the sample immersed for 21 days.
5. Discussion In the present experiment, phosphorylated chitosan films soaked in lime solution were found to be highly susceptible to calcium phosphate growth upon immersion in SBF solution. The phosphorylated films not subjected to the Ca(OH) treatment did not exhibit calcium phosphate growth upon immersion in SBF. During the Ca(OH) treatment, a calcium phosphate precursor phase is expected to be formed all over the chitosan surface which will initiate calcium-deficient HAP growth on subsequent immersion in SBF solution. IR and Ca/P studies showed that the precursor phase would be octacalcium phosphate. During the SBF immersion stage, a calcium phosphate layer having Ca/P ratio less than 1.5 was formed first. Over this layer, secondary nucleation and growth of additional calcium phosphate had occurred resulting in a porous coating. The Ca/P ratio of the coating is found to be very much dependent on the SBF immersion time. Like the results reported in previous publications on biomimetic coatings of calcium phosphates [10], here also the coating was a calcium-deficient apatite and Ca/P ratio increases with increase in soaking time. The mechanism of the above sequence of experiment is suggested as, the Ca(OH) treatment has produced a OCP-type coat ing on the phosphorylated chitosan which facilitate fur-
883
Fig. 6. Thin film XRD pattern of the coating produced after immersion in 1.5;SBF solution for (a) 7 days (b) 21 days.
ther deposition of calcium-deficient apatite on immersion in SBF. The OCP is expected to increase the supersaturation of calcium and phosphate ions further so as to initiate a apatite nucleation. The apatite was substituted by carbonate since SBF is rich in carbonate ions. 6. Conclusion A biomimetic approach has been described for the preparation of calcium-phosphate-coated chitosan films. The coating was porous in nature and may have a variety of uses in the biomedical field. Phosphorylated chitosan films were first soaked in Ca(OH) solution and then immersed in 1.5;SBF for a period of more than 20 days to generate a porous coating of calcium-deficient apatite. The Ca(OH) treatment produces a calcium phosphate precursor coating over the film surface that helps in creating local supersaturation conditions for the precipitation of HAP during the SBF immersion stage. Acknowledgements H.K.V. is grateful to the Science and Technology Agency of Japan for awarding of an STA post-doctoral fellowship to carry out research in Japan. H.K.V. is also grateful to the Director of Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum for granting study leave to use the STA fellowship. References [1] Hench LL, Wilson J. An introduction to bioceramics, Advanced Series in Ceramics, vol. 1. Singapore: World Scientific, 1993.
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H.K. Varma et al. / Biomaterials 20 (1999) 879—884
[2] Abe Y, Kokubo T, Yamamura T. Apatite coating on ceramics, metal and polymers utilizing a biological process. J Mater Sci Mater Med 1990;1:233—8. [3] Dalas E, Kallitis JK, Koutsoukos PG. Crystallization of hydroxyapatite on polymers. Langmuir 1991;7:1822—6. [4] Tretinnikov ON, Kato K, Ikada Y. In vitro hydroxyapatite deposition onto a film surface-grafted with organophosphate polymer. J Biomed Mater Res 1994;28:1365—73. [5] Hato K, Kokubo T, Nakamura T, Yamamura T. Growth of a bone like apatite layer on a substrate by a biomimetic process. J Am Ceram Soc 1995;78(4):1049—53. [6] Mucalo MR, Yokogawa Y, Toriyama M, Suzuki T, Kawamoto Y, Nagata F, Nishizawa K. Growth of calcium phosphate on surface-modified cotton. J Mater Sci Mater Med 1995;6: 597—605.
[7] Yokogawa Y, PazReyes J, Mucalo MR, Toriyama T, Kawamoto Y, Suzuki T, Nishizawa K, Nagata F, Kameyama T. Growth of calcium phosphate on phosphorylated chitin fibers. J Mater Sci Mater Med 1997;8:407—12. [8] Mucalo MR, Yokogawa Y, Toriyama M, Suzuki T, Kawamoto Y, Nagata F, Nishizawa K. Growth of calcium phosphate on ionexchange resins presaturated with calcium or hydrogenphosphate ions: an SEM/EDX and XPS study. J Mater Sci Mater Med 1995;6:409—19. [9] Sakaguchi T, Hirokoshi T, Nakajima A. Adsorption of uranium by chitin phosphate and chitosan phosphate. Agric Biol Chem 1981;45(10):2191—5. [10] Li P, Ohtsuki C, Kokubo T, Nakanishi TK, Soga N, Nakamura T, Yamamuro T. Process of formation of bone like apatite layer on silica gel. J Mater Sci Mater Med 1993;4:127—31.
Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method H.K. Varma* , Y. Yokogawa, F.F. Espinosa, Y. Kawamoto, K. Nishizawa, F. Nagata, T. Kameyama Bioceramic Laboratory, National Industrial Research Institute of Nagoya (NIRIN), Hirate-Cho, Kita-ku, Nagoya 462, Japan Received 26 May 1998; accepted 8 December 1998
Abstract A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energydispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H PO with the P content being 0.1—0.2 wt%. Phosphorylated films soaked in saturated Ca(OH) solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5;SBF for more than 20 days. Phosphorylated films not treated with Ca(OH) did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time. 1999 Elsevier Science Ltd. All rights reserved Keywords: Calcium phosphate; Biomimetic; Chitosan; Coating
1. Introduction Calcium-phosphate-based bioceramics, in the form of powder, granules and coatings are currently used for a number of dental and skeletal prosthetic applications by virtue of their excellent biocompatibility and osteointegration properties [1]. Recently, calcium phosphate materials are being grown biomimetically over different surface-modified substrates in order to gain insight into how to develop biomaterials with tailored surface properties that can initiate calcium phosphate deposition when implanted at bony sites [2—4]. This process has many advantages over conventional coating techniques and can be carried out at ambient reaction conditions.
* Corresponding author. Fax: #471 341 814. Permanent address: Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum 695 012, India.
The nucleation or precipitation of hydroxy apatite over different material surfaces can be achieved by either (a) raising the ionic activity product of calcium phosphate in the solution, thereby stimulating precipitation and creation of apatite nucleation sites [5] or by a (b) surface functionalization method, used to create favourable local conditions that lead to the nucleation and growth of calcium phosphate [6—8]. Deposition of calcium phosphate material over various biopolymeric films has been achieved by biomimetic approach by many researchers with a view to find suitable biomedical applications. These techniques are mainly based on functionalization and subsequent immersion of the substrate material in simulated body fluid solution (SBF). Phosphate group as the functionalization radical was found to be very effective for the deposition of HAP over cotton, chitin, etc. [6, 7]. Prior to immersion in SBF, the phosphorylated substrate is subjected to soaking in saturated Ca(OH) solution. This treatment creates a number of calcium phosphate precursor sites over the surface which will eventually create favourable condition for the nucleation and growth of HAP.
0142-9612/99/$ — see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 9 8 ) 0 0 2 4 3 - 9
880
H.K. Varma et al. / Biomaterials 20 (1999) 879—884
This work describes a method for depositing calcium phosphate mineral over surface-modified chitosan film. Chitosan is a polysaccharide (polymer of 2-amino 2-Dglucose) and is derived by deacetylation of chitin, which is the principal constituent in shells of crabs, lobsters and other crustaceans. It is currently being used in a number of medical applications such as drug encapsulation, fat absorber, and wound dressing materials to name a few. It is non-toxic and biocompatible. It was reported that chitosan phosphate is a very good cation absorber. Ions such as calcium, copper, cadmium, uranium, etc., from solutions were absorbed by phosphorylated chitosan and chitin [9], chitosan phosphate being a better absorbent than chitin phosphate. In the present work, phosphorylated chitosan films soaked in lime were immersed in 1.5;simulated body fluid (SBF) and examined for the calcium phosphate growth over their surfaces at different points in time. 2. Experiments 2.1. Materials All chemicals were supplied by Wako Pure Chemicals Co. and were used without further purification. The growth medium (1.5;SBF) was prepared by procedures reported elsewhere [4], and contained 15 ml of each of the following: 2.74 mol l\ NaCl, 0.06 mol l\ KCl, 0.05 mol l\CaCl , 0.03 mol l\ MgCl , 0.0895 mol l\ NaHCO , 0.02 mol l\ K HPO and 0.01 mol l\ Na SO . These were added to a 200 ml volumetric flask along with 25 ml each of 0.4 mol l\ Tris hydroxy methyl methane amine and 0.36 mol l\ of HCl. The pH of the solution was adjusted to 7.3 by adding a few drops of HCl with the remainder of the volume being distilled water. 2.2. Preparation of chitosan film Chitosan film was made by first dissolving 2 g of commercial chitosan powder in 100 ml of 2% acetic acid solution. The viscous liquid was then poured into glass petri dishes and kept in a vacuum oven at 700 mmHg and 35°C for 6 h to clear all the entrapped gas bubbles. The dishes were then transferred into an air oven and dried at 60°C for 24 h. The chitosan film was easily pealed off when saturated NaOH solution was introduced into the dishes. The wet films were then thoroughly rinsed with distilled water. Thickness of the films ranged between 0.2 and 0.3 mm.
a round-bottomed flask fitted with a condenser, thermometer and nitrogen gas inlet tube. The bottle was then heated to 120°C with the reactants refluxed for 1 h under gentle stirring using a magnetic stirrer. Some foam formation occurred inside the flask during the course of the reaction. After cooling the contents, the films were removed and thoroughly rinsed with distilled water. 2.4. Soaking in saturated Ca(OH)2 solution Some phosphorylated films were suspended in freshly prepared saturated Ca(OH) solution for a period of 8 days. The solution was replaced with fresh Ca(OH) after 3 days. After treatment, the films were thoroughly washed with distilled water. 2.5. Soaking in 1.5;SBF solution Lime soaked, phosphorylated chitosan films were vertically suspended in plastic jars with cotton threads and 50 ml of 1.5;SBF solution was added. The films were freely suspended and did not stick to each other. The jars were then covered with an airtight cap and put into an air oven kept at 36.5°C. The SBF solution was replaced each day. Samples were retrieved after 3, 7, 21 and 30 days of soaking. The retrieved samples were thoroughly rinsed with distilled water and dried at 65°C before performing various characterization studies.
3. Characterization Scanning electron microscopy and EDAX analysis were carried out using a Hitachi S530 scanning electron microscope and a Horiba EMAX 2200 X-ray micro analyzer. Micro-FTIR measurements were done in a Jasco Micro FTIR Jansen Fourier transform infrared spectrometer. Film samples were encased in 1 mm transparent KBr pellets in order to view under the FTIR microscope. The P content of the phosphorylated film and Ca and P content of the Ca(OH) -treated and SBF-treated films were determined using ICP analysis (Nippon Jarrell-Ash ICAP-1000S ICP-AES). Thin film X-ray diffraction studies were recorded using a MXP Material Analysis and Characterization XRD spectrometer using Cu K radiation. ? 4. Results
2.3. Phosphorylation of chitosan films 4.1. Phosphorylated chitosan film The phosphorylation reaction was carried out by a similar procedure reported earlier [9]. Approximately 1 g of chitosan film, 3 g of 98% H PO , 15 g of urea and 30 ml of dimethyl formamide were mixed together in
Untreated and phosphorylated chitosan films were transparent. Figure 1a shows an SEM photograph of the chitosan film. At high magnification, phosphorylated
H.K. Varma et al. / Biomaterials 20 (1999) 879—884
881
Fig. 1. Scanning electron micrograph of chitosan film (a) before and (b) after phosphorylation.
Fig. 3. Scanning electron micrograph of chitosan film after phosphorylation and Ca(OH) soaking for 8 days.
Fig. 2. Micro-FTIR spectrum of (a) chitosan film (b) chitosan film after phosphorylation and (c) after phosphorylation and Ca(OH) soaking for 8 days.
films showed small blisters and bubbles on their film surface (Fig. 1b). EDAX analysis of the above film revealed that the surface is rich in P. As per the ICP analysis of the film, P content is 0.1—0.2 wt%. This value is lower than the one reported for chitosan powder subjected to similar phosphorylation experiments and in that case, the P content was about 6 wt% [9]. Figure 2 shows the micro-FTIR spectrum of the chitosan film before and after phosphorylation. Peaks at 1650 and 1544 cm\ are from the amide bond and the 1000 cm\ is due to the C"O stretching due to the undeacetylated groups of the chitosan (Fig. 2a). The C—O stretch at 899 cm\ is also present. The 1510 and 1370 cm\ are
due to the primary amino group in chitosan. In the pattern for phosphorylated film (Fig. 2b), the PO vibra tion peaks are predominant compared to the existing chitosan peaks. Absorption peaks at 1150 and 1022 cm\ are due to the P—O stretch and 620 cm\ is due to the P—O bend. 4.2. Ca(OH)2-treated phosphorylated chitosan film When phosphorylated chitosan film was soaked in saturated CaO solution for 8 days, a coating of fine particles formed over its surface (Fig. 3), which were determined by EDAX to be calcium phosphate. As per the ICP data obtained for P and Ca, Ca/P ratio was 1.34. The micro-FTIR spectrum (Fig. 2c) showed a broad pattern of poorly crystalline calcium phosphate with 1150 and 620 cm\ peaks ascribed to P—O stretch and bend and are present along with the other chitosan peaks.
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H.K. Varma et al. / Biomaterials 20 (1999) 879—884
4.3. SBF treatment The Ca(OH) -treated phosphorylated chitosan films were immersed in 1.5;SBF solution for different periods of time. Figure 4 shows SEM pictures of the samples retrieved after 3, 7 and 21 days. After 3 days, a single layer of particles of calcium phosphate ((200 nm in size) started depositing over the surface. After 7 days, the surface is almost covered by calcium phosphate particles accompanied by secondary nucleation over the initial layer. This results in a porous aggregate of particles. Within 21 days, the entire surface was covered with
a porous calcium phosphate coating (Fig. 4c). EDAX analysis showed that these particles are calcium phosphate with Ca/P ratio less than 1.67, less than the Ca/P value of pure calcium hydroxy apatite. As the immersion time increases, the Ca/P ratio also increases (Fig. 5). The micro-FTIR spectrum of the phosphate-rich area showed that the coating is poorly crystallized hydroxy apatite. The major peaks at 1100 and 600 cm\ correspond to the PO group. The apatite is a carbonate substituted one as the carbonate peak at 1400 cm\ is quite predominant. Thin-film XRD patterns of the films immersed in SBF for 7 and 21 days are given in Fig. 6. The broad
Fig. 4. Low and high magnification scanning electron micrographs of phosphorylated and Ca(OH) treated chitosan films after immersion in 1.5;SBF solution at 36.5°C for (a and b) 3 days (c and d) 7 days and (e and f ) 21 days.
H.K. Varma et al. / Biomaterials 20 (1999) 879—884
Fig. 5. Plot of Ca/P ratio of the coating produced vs. time of immersion in 1.5;SBF solution for the phosphorylated and Ca(OH) treated chitosan films.
peak of 2h around 32—34° is ascribed to the diffraction of 211, 300 and 202 planes of HAP crystals. Another broad peak around 20° is due to the substrate chitosan film. A more resolved spectrum is obtained for the sample immersed for 21 days.
5. Discussion In the present experiment, phosphorylated chitosan films soaked in lime solution were found to be highly susceptible to calcium phosphate growth upon immersion in SBF solution. The phosphorylated films not subjected to the Ca(OH) treatment did not exhibit calcium phosphate growth upon immersion in SBF. During the Ca(OH) treatment, a calcium phosphate precursor phase is expected to be formed all over the chitosan surface which will initiate calcium-deficient HAP growth on subsequent immersion in SBF solution. IR and Ca/P studies showed that the precursor phase would be octacalcium phosphate. During the SBF immersion stage, a calcium phosphate layer having Ca/P ratio less than 1.5 was formed first. Over this layer, secondary nucleation and growth of additional calcium phosphate had occurred resulting in a porous coating. The Ca/P ratio of the coating is found to be very much dependent on the SBF immersion time. Like the results reported in previous publications on biomimetic coatings of calcium phosphates [10], here also the coating was a calcium-deficient apatite and Ca/P ratio increases with increase in soaking time. The mechanism of the above sequence of experiment is suggested as, the Ca(OH) treatment has produced a OCP-type coat ing on the phosphorylated chitosan which facilitate fur-
883
Fig. 6. Thin film XRD pattern of the coating produced after immersion in 1.5;SBF solution for (a) 7 days (b) 21 days.
ther deposition of calcium-deficient apatite on immersion in SBF. The OCP is expected to increase the supersaturation of calcium and phosphate ions further so as to initiate a apatite nucleation. The apatite was substituted by carbonate since SBF is rich in carbonate ions. 6. Conclusion A biomimetic approach has been described for the preparation of calcium-phosphate-coated chitosan films. The coating was porous in nature and may have a variety of uses in the biomedical field. Phosphorylated chitosan films were first soaked in Ca(OH) solution and then immersed in 1.5;SBF for a period of more than 20 days to generate a porous coating of calcium-deficient apatite. The Ca(OH) treatment produces a calcium phosphate precursor coating over the film surface that helps in creating local supersaturation conditions for the precipitation of HAP during the SBF immersion stage. Acknowledgements H.K.V. is grateful to the Science and Technology Agency of Japan for awarding of an STA post-doctoral fellowship to carry out research in Japan. H.K.V. is also grateful to the Director of Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum for granting study leave to use the STA fellowship. References [1] Hench LL, Wilson J. An introduction to bioceramics, Advanced Series in Ceramics, vol. 1. Singapore: World Scientific, 1993.
884
H.K. Varma et al. / Biomaterials 20 (1999) 879—884
[2] Abe Y, Kokubo T, Yamamura T. Apatite coating on ceramics, metal and polymers utilizing a biological process. J Mater Sci Mater Med 1990;1:233—8. [3] Dalas E, Kallitis JK, Koutsoukos PG. Crystallization of hydroxyapatite on polymers. Langmuir 1991;7:1822—6. [4] Tretinnikov ON, Kato K, Ikada Y. In vitro hydroxyapatite deposition onto a film surface-grafted with organophosphate polymer. J Biomed Mater Res 1994;28:1365—73. [5] Hato K, Kokubo T, Nakamura T, Yamamura T. Growth of a bone like apatite layer on a substrate by a biomimetic process. J Am Ceram Soc 1995;78(4):1049—53. [6] Mucalo MR, Yokogawa Y, Toriyama M, Suzuki T, Kawamoto Y, Nagata F, Nishizawa K. Growth of calcium phosphate on surface-modified cotton. J Mater Sci Mater Med 1995;6: 597—605.
[7] Yokogawa Y, PazReyes J, Mucalo MR, Toriyama T, Kawamoto Y, Suzuki T, Nishizawa K, Nagata F, Kameyama T. Growth of calcium phosphate on phosphorylated chitin fibers. J Mater Sci Mater Med 1997;8:407—12. [8] Mucalo MR, Yokogawa Y, Toriyama M, Suzuki T, Kawamoto Y, Nagata F, Nishizawa K. Growth of calcium phosphate on ionexchange resins presaturated with calcium or hydrogenphosphate ions: an SEM/EDX and XPS study. J Mater Sci Mater Med 1995;6:409—19. [9] Sakaguchi T, Hirokoshi T, Nakajima A. Adsorption of uranium by chitin phosphate and chitosan phosphate. Agric Biol Chem 1981;45(10):2191—5. [10] Li P, Ohtsuki C, Kokubo T, Nakanishi TK, Soga N, Nakamura T, Yamamuro T. Process of formation of bone like apatite layer on silica gel. J Mater Sci Mater Med 1993;4:127—31.