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Wet chemical synthesis of chitosan hydrogel–hydroxyapatite composite membranes for tissue engineering applications
International Journal of Biological Macromolecules 45 (2009) 12–15
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Wet chemical synthesis of chitosan hydrogel–hydroxyapatite composite membranes for tissue engineering applications K. Madhumathi a , K.T. Shalumon a , V.V. Divya Rani a , H. Tamura b , T. Furuike b , N. Selvamurugan a , S.V. Nair a , R. Jayakumar a,∗ a
Amrita Centre for Nanosciences, Amrita Institute of Medical Sciences and Research Centre, Amrita Viswa Vidyapeetham University, Kochi 682 026, India Faculty of Chemistry, Materials and Bioengineering & High Technology Research Centre, Kansai University, Osaka 564-8680, Japan b
a r t i c l e
i n f o
Article history: Received 6 March 2009 Received in revised form 23 March 2009 Accepted 25 March 2009 Available online 2 April 2009 Keywords: Chitosan hydrogel membranes Composite membranes Tissue engineering Hydroxyapatite Biomaterials
a b s t r a c t Chitosan, a deacetylated derivative of chitin is a commonly studied biomaterial for tissue-engineering applications due to its biocompatibility, biodegradability, low toxicity, antibacterial activity, wound healing ability and haemostatic properties. However, chitosan has poor mechanical strength due to which its applications in orthopedics are limited. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has mechanical strength and osteoconductive property. In this work, HAp was deposited on the surface of chitosan hydrogel membranes by a wet chemical synthesis method by alternatively soaking the membranes in CaCl2 (pH 7.4) and Na2 HPO4 solutions for different time intervals. These chitosan hydrogel–HAp membranes were characterized using SEM, AFM, EDS, FT-IR and XRD analyses. MTT assay was done to evaluate the biocompatibility of these membranes using MG-63 osteosarcoma cells. The biocompatibility studies suggest that chitosan hydrogel–HAp composite membranes can be useful for tissue-engineering applications. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering is the technology of remodeling of living organisms in vitro and involves architecture of artificial cellular scaffolds, which mimic extracellular matrix. Recently biopolymeric materials have been used for tissue engineering and other biomedical applications. As a biomedical material, calcium phosphate has bioactive and osteoconductive properties [1]. The most widely occurring biological calcium phosphate is HAp. It is the main inorganic component of bones and teeth having biocompatibility for biomedical applications. Several investigators have studied implant materials for replacement of hard tissues, such as titanium and titanium alloy coated with HAp [2,3]. But, there is little research on implants materials for soft tissue adhesion. Therefore we have developed the chitosan hydrogel–Hap composites as novel implantable materials that contact soft tissue. Chitosan is a commonly studied biopolymer obtained by partial deacetylation of chitin, a natural polymer present in shells of crustaceans, arthropods and some fungi. It is a copolymer of glucosamine and N-acetylglucosamine connected by a ˇ (1–4) linkage
∗ Corresponding author. Tel.: +91 484 2801234; fax: +91 484 2802020. E-mail addresses: [email protected], [email protected] (R. Jayakumar). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.03.011
[4–8]. They are widely used as biomaterials due to excellent biocompatibility, biodegradability, low toxicity, wound healing ability, haemostatic property as well as antimicrobial activity [8–11]. However, chitosan has poor mechanical strength, which limits its use as a tissue supporting material in bone tissue engineering [12,13]. Also, chitosan is not sufficiently osteogenic to induce the desired rapid bone regeneration during initial healing period [14,15]. To improve the mechanical properties as well as osteoconductivity of chitosan, its blending with HAp has been widely investigated [16–19]. HAp [Ca10 (PO4 )6 (OH)2 ] is widely used to enhance the mechanical, bioactive and osteoconductive properties of biopolymers [20–24]. HAp is a biomimetic material, found as an inorganic component of bone and teeth. HAp is also a highly osteoconductive material facilitating better bonding of the biomaterial with bone [25]. Biopolymer–HAp composites have been synthesized by many methods like blending [26], biomimetic process using simulated body fluid (SBF) [27–29], in situ precipitation [18,30] and electrochemical deposition [31,32]. These processes are either complex or time consuming. A relatively simple way of preparing polymer–HAp composite is the wet chemical synthesis method where the polymer matrices are alternatively soaked in CaCl2 (pH 7.4) and Na2 HPO4 solutions [33]. In this paper, we describe the preparation, characterization and biocompatibility studies of chitosan hydrogel–HAp composite membranes in detail.
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2.4. Biocompatibility studies Biocompatibility of the chitosan hydrogel–HAp composite membranes was assessed using MTT assay. Tissue culture well was taken as the control. Chitosan hydrogel–HAp membranes were placed in a 96-well plates and MG-63 Osteosarcoma cells were seeded on membranes with a density of 1000 cells/well. MTT [3(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium] assay was used to quantify the cells grown on the membranes. 5 mg of MTT (Sigma) was dissolved in 1 ml of PBS and filter sterilized. 10 l of the MTT solution was further diluted to 100 l with 90 l of serumfree phenol red free minimum essential medium. The membranes kept in 96 well plates were incubated with 100 l of the above solution to form formazan crystals by mitochondrial dehydrogenases. After 24 h of incubation at 37 ◦ C, 100 l of the solubilization solution (10% Triton X-100, 0.1N HCl and isopropanol) was added in each well plate to dissolve the formazan crystals. The optical density of the solution was measured at a wavelength of 570 nm using an Elisa plate reader [Beckmann Coulter DTX 880]. Duplicate samples were analyzed for each experiment. 2.5. Characterization
Fig. 1. Experimental techniques of an alternate soaking process of chitosan gel membranes.
2. Experimental 2.1. Materials Chitosan (degree of deacetylation-85%) was received from KYOWA TECNOS Co. Ltd. Calcium Chloride (CaCl2 -purity-99%) was purchased from SIGMA and disodium hydrogen phosphate (Na2 HPO4 -purity-99.5%) was purchased from S.D FINECHEM. Tris(hydroxymethyl)aminomethane (Tris) (purity-99%) and hydrochloric acid (HCL) were purchased from Qualigens. 2.2. Preparation of chitosan hydrogel membranes Chitosan (20 g) was dissolved in 1000 ml of 1% acetic acid solution. The pH of chitosan solution was increased to 7.0 by the addition of 1 M NaOH. When the pH of the solution reached to 7, chitosan hydrogel was formed. The chitosan hydrogel thus obtained was dialyzed for 2 days in distilled water for purification. The purified hydrogel was dried at 1 Ton pressure at room temperature for a day to obtain the gel membranes. After that the membranes were cut into disc form (1 cm in diameter and 1 mm in thickness) for the preparation of chitosan hydrogel–HAp composite membranes. 2.3. Preparation of chitosan hydrogel–HAp composite membranes Chitosan hydrogel membranes in the form of discs were immersed in 10 ml of CaCl2 solution (200 mM) (pH 7.4 adjusted with Tris–HCl) for 2 h at 37 ◦ C. Then it was taken out and excess moisture was removed by blotting with tissue paper. Following this, the membranes were soaked in 10 ml of Na2 HPO4 solution (120 mM) for 2 h at 37 ◦ C. This comprised one cycle and for our experiments the number of cycles carried out was up to 5. Finally the membranes were washed with distilled water and air dried at 37 ◦ C. The schematic diagram of alternate soaking process is shown in Fig. 1.
The surface deposition of HAp was investigated using Scanning Electron Microscopy (SEM) (JEOL Ltd., JEOLJSM-6490LA) with Energy Dispersive Spectrum (EDS). The X-ray diffraction studies (XRD) of composite membranes were carried out using an XRD [Rigaku Dmax-C] fitted with Cu K␣ ( = 1.541 Å) ranging from 20 to 80◦ at 0.05◦ /min. Phase identification was carried out with the help of standard JCPDS database. The composite membranes were also characterized using PerkinElmer Co., SPECTRUM RX1, FT-IR. 3. Results and discussion 3.1. Preparation of chitosan hydrogel–HAp composite membranes Hap was formed on swollen chitosan hydrogel membranes by alternate soaking process. The reaction involves salt formation between calcium and phosphate ions. In this study, the hydrogel membranes were first immersed in CaCl2 solution. The hydrogel membranes were washed with distilled water to get rid of excess calcium ions before soaking in Na2 HPO4 solution. The calcium ions displace free water molecules, and repulse each other to give enough space to react with phosphate ions. The resulting HAp deposits seemed to function as nucleating particles to induce calcium and phosphate ions to enter the swollen hydrogel membranes for the next alternate cycle. The HAp was completely dispersed throughout the swollen hydrogel membranes after five cycles. 3.2. SEM and AFM studies Fig. 2 shows the presence of HAp crystals on the surface of chitosan hydrogel membranes after alternate soaking. There was increased deposition of HAp crystals with increasing number of immersion cycles. This was also confirmed by using AFM studies. The deposition of HAp was increased from nano- to microlevel (from 356 nm to 2.17 m), when the immersion cycles were increased. 3.3. EDS studies The deposition of HAp was also confirmed by EDS analysis. The EDS obtained had the Ca/P ratio ranging from 1.68 to 1.87 in all the cycles. The Ca/P ratios corresponded to that of HAp [34]. Fig. 3 shows the EDS spectrum of the membranes soaked at three cycles.
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K. Madhumathi et al. / International Journal of Biological Macromolecules 45 (2009) 12–15
Fig. 2. SEM images of chitosan gel membranes: (a) control, (b) after one cycle, (c) after three cycles and (d) after five cycles of soaking in CaCl2 (pH 7.4) and Na2 HPO4 solutions.
3.4. FT-IR studies Fig. 4 shows the FT-IR spectra of chitosan hydrogel–HAp composite membranes. The peaks at 602 and 575 cm−1 corresponded to 4 PO4 vibrations. The peak at 1034 cm−1 was assigned to 3 PO4 vibrations. The peak at 3440 cm−1 was due to hydroxyl group. The peak at 1432 cm−1 was due to calcium phosphate [34–36]. The peak at 1650 cm−1 decreased in the mineralized membranes, indicating the interaction of the amine on the chitosan and phosphates in the solution [37]. The intensity of peaks increased with increased number of immersion cycles as shown in Fig. 4. This result also correlated with our AFM findings of increased deposition of HAp with increased soaking time or immersion cycles.
branes, sharp diffraction characteristic peaks occurred at 28.4◦ and 23.8◦ , implying the presence of HAp in the composite membranes [38]. The broad peak around at 2 = 31.8◦ was a summed contribution of the (2 1 1), (1 1 2) and (3 0 0) lattice planes of HAp [39]. The other peaks at 26.0◦ , 32.9◦ , 39.8◦ and 46.7◦ are also the characteristic peaks of HAp [17,31,34]. The above peaks also corresponded to (2 1 1) HAp (JCPDS #09-0432). The peak around at 2 = 20◦ was due to chitosan. The XRD analysis in combination with FT-IR analysis clearly indicated the formation of HAp in the composite membranes. 3.6. Biocompatibility studies
Fig. 5 shows the XRD patterns of the chitosan hydrogel–HAp composite membranes. In chitosan hydrogel–HAp composite mem-
Biocompatibility of the chitosan hydrogel–HAp composite membranes was analyzed using MTT assay as described earlier. Here, in our study we used three different composite membranes subjected to varying HAp deposition cycles viz; one cycle, three cycles and five cycles. After 24 h of incubation no significant
Fig. 3. EDS spectrum of chitosan gel membrane (after three cycles).
Fig. 4. FT-IR spectra of chitosan gel membranes: (a) control, (b) after one cycle, (c) after three cycles and (d) after five cycles of soaking in CaCl2 (pH 7.4) and Na2 HPO4 solutions.
3.5. XRD studies
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the preparation of biocompatible chitosan hydrogel–HAp composite membranes. Acknowledgments This work was supported by the Department of Science and Technology, Government of India, under the Nanoscience and Nanotechnology Initiative program monitored by Dr. C. N. R. Rao. References
Fig. 5. XRD spectrum of chitosan gel membranes (a) after one cycle, (b) after three cycles and (c) after five cycles of soaking in CaCl2 (pH 7.4) and Na2 HPO4 solutions.
Fig. 6. Graph showing the biocompatability of chitosan gel membranes compared to control (TCP) after 24 h of incubation using MTT assay.
difference in cell growth was observed among the composite membranes. Similarly all the membranes shows excellent biocompatibility compared to the control (Fig. 6). 4. Conclusions Chitosan hydrogel–HAp composite membranes were prepared by the wet synthesis method by alternate soaking of membranes in CaCl2 (pH 7.4) and Na2 HPO4 solutions for 1, 3 and 5 cycles. The prepared hydrogel composite membranes were characterized using SEM, AFM, EDS, FT-IR and XRD. The results showed that HAp deposition occurred on the surface of chitosan hydrogel membranes within short period of time i.e. (20 h). Excellent biocompatibility results propose that these membranes may have potential applications in tissue-engineering field. Thus, these results suggest that alternate soaking method may be a simple method for
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Wet chemical synthesis of chitosan hydrogel–hydroxyapatite composite membranes for tissue engineering applications K. Madhumathi a , K.T. Shalumon a , V.V. Divya Rani a , H. Tamura b , T. Furuike b , N. Selvamurugan a , S.V. Nair a , R. Jayakumar a,∗ a
Amrita Centre for Nanosciences, Amrita Institute of Medical Sciences and Research Centre, Amrita Viswa Vidyapeetham University, Kochi 682 026, India Faculty of Chemistry, Materials and Bioengineering & High Technology Research Centre, Kansai University, Osaka 564-8680, Japan b
a r t i c l e
i n f o
Article history: Received 6 March 2009 Received in revised form 23 March 2009 Accepted 25 March 2009 Available online 2 April 2009 Keywords: Chitosan hydrogel membranes Composite membranes Tissue engineering Hydroxyapatite Biomaterials
a b s t r a c t Chitosan, a deacetylated derivative of chitin is a commonly studied biomaterial for tissue-engineering applications due to its biocompatibility, biodegradability, low toxicity, antibacterial activity, wound healing ability and haemostatic properties. However, chitosan has poor mechanical strength due to which its applications in orthopedics are limited. Hydroxyapatite (HAp) is a natural inorganic component of bone and teeth and has mechanical strength and osteoconductive property. In this work, HAp was deposited on the surface of chitosan hydrogel membranes by a wet chemical synthesis method by alternatively soaking the membranes in CaCl2 (pH 7.4) and Na2 HPO4 solutions for different time intervals. These chitosan hydrogel–HAp membranes were characterized using SEM, AFM, EDS, FT-IR and XRD analyses. MTT assay was done to evaluate the biocompatibility of these membranes using MG-63 osteosarcoma cells. The biocompatibility studies suggest that chitosan hydrogel–HAp composite membranes can be useful for tissue-engineering applications. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering is the technology of remodeling of living organisms in vitro and involves architecture of artificial cellular scaffolds, which mimic extracellular matrix. Recently biopolymeric materials have been used for tissue engineering and other biomedical applications. As a biomedical material, calcium phosphate has bioactive and osteoconductive properties [1]. The most widely occurring biological calcium phosphate is HAp. It is the main inorganic component of bones and teeth having biocompatibility for biomedical applications. Several investigators have studied implant materials for replacement of hard tissues, such as titanium and titanium alloy coated with HAp [2,3]. But, there is little research on implants materials for soft tissue adhesion. Therefore we have developed the chitosan hydrogel–Hap composites as novel implantable materials that contact soft tissue. Chitosan is a commonly studied biopolymer obtained by partial deacetylation of chitin, a natural polymer present in shells of crustaceans, arthropods and some fungi. It is a copolymer of glucosamine and N-acetylglucosamine connected by a ˇ (1–4) linkage
∗ Corresponding author. Tel.: +91 484 2801234; fax: +91 484 2802020. E-mail addresses: [email protected], [email protected] (R. Jayakumar). 0141-8130/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2009.03.011
[4–8]. They are widely used as biomaterials due to excellent biocompatibility, biodegradability, low toxicity, wound healing ability, haemostatic property as well as antimicrobial activity [8–11]. However, chitosan has poor mechanical strength, which limits its use as a tissue supporting material in bone tissue engineering [12,13]. Also, chitosan is not sufficiently osteogenic to induce the desired rapid bone regeneration during initial healing period [14,15]. To improve the mechanical properties as well as osteoconductivity of chitosan, its blending with HAp has been widely investigated [16–19]. HAp [Ca10 (PO4 )6 (OH)2 ] is widely used to enhance the mechanical, bioactive and osteoconductive properties of biopolymers [20–24]. HAp is a biomimetic material, found as an inorganic component of bone and teeth. HAp is also a highly osteoconductive material facilitating better bonding of the biomaterial with bone [25]. Biopolymer–HAp composites have been synthesized by many methods like blending [26], biomimetic process using simulated body fluid (SBF) [27–29], in situ precipitation [18,30] and electrochemical deposition [31,32]. These processes are either complex or time consuming. A relatively simple way of preparing polymer–HAp composite is the wet chemical synthesis method where the polymer matrices are alternatively soaked in CaCl2 (pH 7.4) and Na2 HPO4 solutions [33]. In this paper, we describe the preparation, characterization and biocompatibility studies of chitosan hydrogel–HAp composite membranes in detail.
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2.4. Biocompatibility studies Biocompatibility of the chitosan hydrogel–HAp composite membranes was assessed using MTT assay. Tissue culture well was taken as the control. Chitosan hydrogel–HAp membranes were placed in a 96-well plates and MG-63 Osteosarcoma cells were seeded on membranes with a density of 1000 cells/well. MTT [3(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium] assay was used to quantify the cells grown on the membranes. 5 mg of MTT (Sigma) was dissolved in 1 ml of PBS and filter sterilized. 10 l of the MTT solution was further diluted to 100 l with 90 l of serumfree phenol red free minimum essential medium. The membranes kept in 96 well plates were incubated with 100 l of the above solution to form formazan crystals by mitochondrial dehydrogenases. After 24 h of incubation at 37 ◦ C, 100 l of the solubilization solution (10% Triton X-100, 0.1N HCl and isopropanol) was added in each well plate to dissolve the formazan crystals. The optical density of the solution was measured at a wavelength of 570 nm using an Elisa plate reader [Beckmann Coulter DTX 880]. Duplicate samples were analyzed for each experiment. 2.5. Characterization
Fig. 1. Experimental techniques of an alternate soaking process of chitosan gel membranes.
2. Experimental 2.1. Materials Chitosan (degree of deacetylation-85%) was received from KYOWA TECNOS Co. Ltd. Calcium Chloride (CaCl2 -purity-99%) was purchased from SIGMA and disodium hydrogen phosphate (Na2 HPO4 -purity-99.5%) was purchased from S.D FINECHEM. Tris(hydroxymethyl)aminomethane (Tris) (purity-99%) and hydrochloric acid (HCL) were purchased from Qualigens. 2.2. Preparation of chitosan hydrogel membranes Chitosan (20 g) was dissolved in 1000 ml of 1% acetic acid solution. The pH of chitosan solution was increased to 7.0 by the addition of 1 M NaOH. When the pH of the solution reached to 7, chitosan hydrogel was formed. The chitosan hydrogel thus obtained was dialyzed for 2 days in distilled water for purification. The purified hydrogel was dried at 1 Ton pressure at room temperature for a day to obtain the gel membranes. After that the membranes were cut into disc form (1 cm in diameter and 1 mm in thickness) for the preparation of chitosan hydrogel–HAp composite membranes. 2.3. Preparation of chitosan hydrogel–HAp composite membranes Chitosan hydrogel membranes in the form of discs were immersed in 10 ml of CaCl2 solution (200 mM) (pH 7.4 adjusted with Tris–HCl) for 2 h at 37 ◦ C. Then it was taken out and excess moisture was removed by blotting with tissue paper. Following this, the membranes were soaked in 10 ml of Na2 HPO4 solution (120 mM) for 2 h at 37 ◦ C. This comprised one cycle and for our experiments the number of cycles carried out was up to 5. Finally the membranes were washed with distilled water and air dried at 37 ◦ C. The schematic diagram of alternate soaking process is shown in Fig. 1.
The surface deposition of HAp was investigated using Scanning Electron Microscopy (SEM) (JEOL Ltd., JEOLJSM-6490LA) with Energy Dispersive Spectrum (EDS). The X-ray diffraction studies (XRD) of composite membranes were carried out using an XRD [Rigaku Dmax-C] fitted with Cu K␣ ( = 1.541 Å) ranging from 20 to 80◦ at 0.05◦ /min. Phase identification was carried out with the help of standard JCPDS database. The composite membranes were also characterized using PerkinElmer Co., SPECTRUM RX1, FT-IR. 3. Results and discussion 3.1. Preparation of chitosan hydrogel–HAp composite membranes Hap was formed on swollen chitosan hydrogel membranes by alternate soaking process. The reaction involves salt formation between calcium and phosphate ions. In this study, the hydrogel membranes were first immersed in CaCl2 solution. The hydrogel membranes were washed with distilled water to get rid of excess calcium ions before soaking in Na2 HPO4 solution. The calcium ions displace free water molecules, and repulse each other to give enough space to react with phosphate ions. The resulting HAp deposits seemed to function as nucleating particles to induce calcium and phosphate ions to enter the swollen hydrogel membranes for the next alternate cycle. The HAp was completely dispersed throughout the swollen hydrogel membranes after five cycles. 3.2. SEM and AFM studies Fig. 2 shows the presence of HAp crystals on the surface of chitosan hydrogel membranes after alternate soaking. There was increased deposition of HAp crystals with increasing number of immersion cycles. This was also confirmed by using AFM studies. The deposition of HAp was increased from nano- to microlevel (from 356 nm to 2.17 m), when the immersion cycles were increased. 3.3. EDS studies The deposition of HAp was also confirmed by EDS analysis. The EDS obtained had the Ca/P ratio ranging from 1.68 to 1.87 in all the cycles. The Ca/P ratios corresponded to that of HAp [34]. Fig. 3 shows the EDS spectrum of the membranes soaked at three cycles.
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Fig. 2. SEM images of chitosan gel membranes: (a) control, (b) after one cycle, (c) after three cycles and (d) after five cycles of soaking in CaCl2 (pH 7.4) and Na2 HPO4 solutions.
3.4. FT-IR studies Fig. 4 shows the FT-IR spectra of chitosan hydrogel–HAp composite membranes. The peaks at 602 and 575 cm−1 corresponded to 4 PO4 vibrations. The peak at 1034 cm−1 was assigned to 3 PO4 vibrations. The peak at 3440 cm−1 was due to hydroxyl group. The peak at 1432 cm−1 was due to calcium phosphate [34–36]. The peak at 1650 cm−1 decreased in the mineralized membranes, indicating the interaction of the amine on the chitosan and phosphates in the solution [37]. The intensity of peaks increased with increased number of immersion cycles as shown in Fig. 4. This result also correlated with our AFM findings of increased deposition of HAp with increased soaking time or immersion cycles.
branes, sharp diffraction characteristic peaks occurred at 28.4◦ and 23.8◦ , implying the presence of HAp in the composite membranes [38]. The broad peak around at 2 = 31.8◦ was a summed contribution of the (2 1 1), (1 1 2) and (3 0 0) lattice planes of HAp [39]. The other peaks at 26.0◦ , 32.9◦ , 39.8◦ and 46.7◦ are also the characteristic peaks of HAp [17,31,34]. The above peaks also corresponded to (2 1 1) HAp (JCPDS #09-0432). The peak around at 2 = 20◦ was due to chitosan. The XRD analysis in combination with FT-IR analysis clearly indicated the formation of HAp in the composite membranes. 3.6. Biocompatibility studies
Fig. 5 shows the XRD patterns of the chitosan hydrogel–HAp composite membranes. In chitosan hydrogel–HAp composite mem-
Biocompatibility of the chitosan hydrogel–HAp composite membranes was analyzed using MTT assay as described earlier. Here, in our study we used three different composite membranes subjected to varying HAp deposition cycles viz; one cycle, three cycles and five cycles. After 24 h of incubation no significant
Fig. 3. EDS spectrum of chitosan gel membrane (after three cycles).
Fig. 4. FT-IR spectra of chitosan gel membranes: (a) control, (b) after one cycle, (c) after three cycles and (d) after five cycles of soaking in CaCl2 (pH 7.4) and Na2 HPO4 solutions.
3.5. XRD studies
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the preparation of biocompatible chitosan hydrogel–HAp composite membranes. Acknowledgments This work was supported by the Department of Science and Technology, Government of India, under the Nanoscience and Nanotechnology Initiative program monitored by Dr. C. N. R. Rao. References
Fig. 5. XRD spectrum of chitosan gel membranes (a) after one cycle, (b) after three cycles and (c) after five cycles of soaking in CaCl2 (pH 7.4) and Na2 HPO4 solutions.
Fig. 6. Graph showing the biocompatability of chitosan gel membranes compared to control (TCP) after 24 h of incubation using MTT assay.
difference in cell growth was observed among the composite membranes. Similarly all the membranes shows excellent biocompatibility compared to the control (Fig. 6). 4. Conclusions Chitosan hydrogel–HAp composite membranes were prepared by the wet synthesis method by alternate soaking of membranes in CaCl2 (pH 7.4) and Na2 HPO4 solutions for 1, 3 and 5 cycles. The prepared hydrogel composite membranes were characterized using SEM, AFM, EDS, FT-IR and XRD. The results showed that HAp deposition occurred on the surface of chitosan hydrogel membranes within short period of time i.e. (20 h). Excellent biocompatibility results propose that these membranes may have potential applications in tissue-engineering field. Thus, these results suggest that alternate soaking method may be a simple method for
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