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chitosan cross-linking composite membrane intended for tissue engineering
International Journal of Biological Macromolecules 50 (2012) 43–49
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation and characterization of nano-hydroxyapatite/chitosan cross-linking composite membrane intended for tissue engineering XingYi Li a,∗ , KaiHui Nan a , Shuai Shi b , Hao Chen a,∗ a b
Institute of Biomedical Engineering, School of Ophthalmology & Optometry and Eye hospital, Wenzhou Medical College, 270 Xueyuan Road, Wenzhou 325027, China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, No. 1, Keyuan 4th Road, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 5 September 2011 Accepted 24 September 2011 Available online 1 October 2011 Keywords: Chitosan Nano-hydroxyapatite In vitro cytotoxicity Composite membrane Cross-linking
a b s t r a c t In this paper, a series of nano-hydroxyapatite(n-HA)/chitosan cross-linking composite membranes (nHA; 0, 5, 10, 15, 20 and 30 wt%) were successfully developed by a simple casting/solvent evaporation method. n-HA with size about 20 nm in vertical diameter and about 100 nm in horizontal diameter was successfully synthesized by a hydro-thermal precipitation method, and then dispersed into chitosan/genipin solution with the aid of continuous ultrasound to develop n-HA/chitosan cross-linking composite membranes. The detailed characterizations including Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water adsorption and tensile test were performed. With the analysis of FTIR spectra and TGA spectra, it suggested that there was existence of possible interactions between polymer and n-HA. Meanwhile, the n-HA content was greatly effected on the morphology as well as the tensile property of composite membrane. In vitro cytotoxicity test suggested that the developed n-HA/chitosan cross-linking composite membrane was non-cytotoxicity against L929 cells after 24 h’s incubation might be suitable for further in vivo application. © 2011 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. In past several decades, polymer blend membrane/film has been widely used for tissue engineering and other biomedical applications [1,2]. As well known to us, an ideal membrane for tissue engineering is strongly desired to have good mechanical property, swelling behavior, biocompatibility, bioactivity, and so on [3]. Nowadays, some non-biodegradable membranes, such as expanded polytetrafluoroethylene (e-PTFE) and so on have been clinically employed as barrier membranes for guided bone regeneration [4,5]. However, as the non-biodegradable/absorbable matrix, the implant membrane must be removed out from the body by a second surgical procedure after the healing of bone defect, which will result in the risk of tissue morbidity. Therefore, more and more attention have been oriented to the biodegradable/absorbable membranes made from the
∗ Corresponding authors at: School of Ophthalmology & Optometry and Eye hospital, Wenzhou Medical College, 270 Xueyuan Road, China. Tel.: +86 577 88833806; fax: +86 577 88833806. E-mail addresses: lixingyi [email protected] (X. Li), [email protected] (H. Chen). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.09.021
natural/synthetic polymers such as polylactic acid, chitosan, alginate, and collagen [6–8]. Among these biodegradable/absorbable membranes, chitosan membrane/film has gained considerable attention because of its favorable properties such as low-toxicity, good biocompatibility, biodegradability, mucoadhesive, and easy fabrication [9]. As the cationic polysaccharide in nature, chitosan is composed of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, only dissolve in some acidic solution as pH value was below 6 [10,11]. In past three decades, it has been studied intensively for its applications in pharmaceutical science, cosmetics, biomedical fields, agriculture, and food industries as well as in sewage treatment, paper industry [12–15]. Recently, chitosan and its derivatives have been successfully fabricated into films/membranes for various medical applications such as tissue engineering, wound healing and so on [12,16]. However, some shortcoming made chitosan membrane/film unwelcome in medical application due to its high sensitivity to water as well as only dissolve in acidic solution during the fabrication process. In order to exploit the unique properties of this versatile cationic polysaccharide, several attempts are being made to cross-linking chitosan membrane/film to modulate its hydrophobic–hydrophilic balance and mechanical property, realizing its full potential applications [17,18]. Genipin, as a water soluble bi-functional cross-linking reagent, has been widely used to cross-linking chitosan matrix, proteins and amine in general [19]. Meanwhile, the resulting crosslinked complexes are
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non-cytotoxicity for the animal and human cells as so far examined. The safety and the beneficial actions of genipin has been used a number of research projects in the areas of the therapies of diabetes, periodontitis, cataract, hepatic dysfunction, as well as in wound repair and nerve regeneration [19,20]. Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HA) has been widely used in tissue engineering fields due to its shape was needle-like and its structure and composition similar to minerals of natural bones [21–23]. It not only plays a primary role in improving the mechanical properties of the nanocomposites, but also provides a favorable environment for osteoconduction, protein adhesion, and osteoblast proliferation. Biopolymer/n-HA composites membranes/films have been prepared by many methods like blending, biomimetic process using simulated body fluid (SBF), in situ precipitation and electrochemical deposition [24,25]. Among these methods, the blending is a simple method to develop the n-HA/chitosan cross-linking composite film, that is to say, n-HA/chitosan cross-linking composite membrane could be obtained by simple dispersing n-HA into chitosan–genipin solution. The cross-linking degree of membranes as well as the mechanical property could be modulated by changing the genipin concentration in the formulation, and the unreactive genipin could be extracted by immersion of membrane in phosphate buffered solution (PBS) overnight. Furthermore, the application of genipin to cross-link the composite membrane could significantly improve the hydrophobic property of the composite membrane. The obtained series of n-HA loaded chitosan composite membranes were carefully characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water adsorption and tensile test before its further in vivo application.
2. Materials and methods 2.1. Materials Chitosan (92% degree of deacetylation (DD)) with molecular weight about 200 kDa was supplied by Sigma–Aldrich (USA). Genipin was purchased from Shanghai Y-S Biotechnology Co. LTD (China). Ca(NO3 )·4H2 O and (NH4 )2 HPO4 ·3H2 O were obtained from WenZhou Chemical Reagents Co. LTD (China). All other chemicals used in this paper were analytical grade. Ultrapure water from Milli-Q water system was used to prepare the aqueous solutions.
2.2. Preparation and characterization of nano-hydroxyapatite (n-HA) Pure nano-hydroxyapatite (n-HA) powder was synthesized by a hydro-thermal precipitation method as previous report with little modification [16]. Ca(NO3 )·4H2 O and (NH4 )2 HPO4 ·3H2 O were used as starting materials, and the reaction was performed according to the following reaction: Ca(NO3 )·4H2 O + (NH4 )2 HPO4 ·3H2 O → Ca10 (PO4 )6 (OH)2 The pH value of reaction system was adjusted to 10 by addition of ammonium hydroxide (NH3 ·H2 O) solution. The obtained raw HA precipitation was first treated at 80 ◦ C under normal atmospheric pressure for 2 h. And then the precipitation was centrifuged and fully washed to neutrality by distilled water and anhydrous alcohol. Finally, the obtained n-HA slurry was sintered at 1000 ◦ C for 2 h, and then ground into powder. The morphological characterization of nHA was observed with a transmission electron microscopy (TEM) (H-6009IV, Hitachi, Japan).
Table 1 n-HA/chitosan cross-linking composite membrane made from various n-HA contents. Samples
Chitosan concentration (mg/ml)
n-HA content (%)
Genipin concentration (mM)
S-1 S-2 S-3 S-4 S-5 S-6
20 20 20 20 20 20
0 5 10 15 20 30
0.5 0.5 0.5 0.5 0.5 0.5
2.3. Preparation and characterization of n-HA/chitosan cross-linking composite membrane 2.3.1. Preparation of n-HA/chitosan cross-linking composite membrane The casting/solvent evaporation technology was employed to develop the n-HA/chitosan cross-linking composite membranes [25,26]. Initially, chitosan (4 g) was dissolved in 1% acetic acid solution (200 ml) under magnetic stirring for 48 h at room temperature. After that, genipin was added to the above solution with the final concentration at 0.5 mM. Subsequently, the obtained n-HA powder regardless of various weight ratios of n-HA/chitosan, was added to the above chitosan–genipin solution with the continuous ultrasound to obtain the homogeneous n-HA/chitosan–genipin suspension. Finally, 20 ml n-HA/chitosan–genipin suspension was poured into a glass Petri dish (d = 6 cm) and dried at 37 ◦ C for 2 days to obtain the n-HA/chitosan cross-linking composite membranes, as shown in Table 1. In order to remove the resident acetic acid and unreactive genipin, the obtained n-HA/chitosan cross-linking composite membranes were immersed into phosphate buffered saline (PBS) solution for about 6 h, and then washed with distilled water twice to neutral. All membranes were re-dried at 37 ◦ C for 2 days before the further characterization and application. 2.3.2. Fourier transform infrared spectroscopy (FTIR) measurement FTIR (KBr pellet) spectra were performed at room temperature using a NICOLET 200SXV Infrared Spectrophotometer (USA). The characteristic absorption bands of the composite membranes were detected at wavenumbers ranging from 500 to 4000 cm−1 . 2.3.3. Crystallographic assay X-ray diffraction spectrometry was obtained by using X-ray Diffractometer (DX-2000, DanDong Fangyuan Instrument Company, China) using CuKa radiation. The relative intensities were recorded within the range of 10–60◦ (2) at a scanning rate of 4◦ min−1 . 2.3.4. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed by a thermogravimetric analyzer (TA 2910, DuPont, USA) under a steady flow of nitrogen atmosphere at a heating rate of 10 ◦ C/min in the range of 20–600 ◦ C. 2.3.5. Morphological analysis The morphological characterization of composite membrane was performed by scanning electron microscopy (JSM-5900LV, JEOL, Japan). Composite membrane samples were placed at cabinet drier for 24 h before the observation. The upper and bottom surface of composite membrane was observed respectively.
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2.3.6. Tensile test Rectangular specimens of the n-HA/chitosan crosslinking composite membranes with dimensions of 40 mm × 5 mm × 0.06–0.10 mm were tested by a universal mechanical testing instrument (Instron-5567, Instron Corp., USA) at room temperature and relative humidity of 50%. The tensile strength and elongation were evaluated at a displacement rate of 20 mm/min with 20-mm gauge length. All results were mean values of five specimens.
2.4. Water adsorption studies Water adsorption of n-HA/chitosan cross-linking composite membranes was detected by weighing the membranes pieces before and after placing in 20 ml distilled water solution. Each membrane was divided in portions of 1 cm2 (1 cm × 1 cm), weighed and placed in distilled water solution for periodical study (5, 10, 20, 40, 60 and 90 min) as described by Rodrigues et al. [27]. At specific time intervals, the membranes were taken from the medium and weighed after removal of the surplus surface water using filter paper. The water absorption were calculated by following: Water adsorption (%) =
W90 − W0 × 100 W0
(1)
where W90 is the weight of wet membrane at 90 min and W0 is the original membrane weight at dry state, respectively. Here, the water absorption of composite membrane after immersed in distilled water solution for 90 min was defined as equilibriumswelling ratio. This experiment was performed in triplicate.
2.5. In vitro cytotoxicity test According to the previous studies, MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) test was used to evaluate in vitro cytotoxicity of n-HA/chitosan cross-linking composite membranes [28,29]. The mouse fibroblastic-like cells (L929 cell) were cultured with DMEM medium at 37 ◦ C and 5% CO2 . Due to the insoluble of composite membrane in water solution, the leachables of polymeric component from the n-HA/chitosan cross-linking composite membrane was employed to evaluate the cytotoxicity of composite membrane. Initially, cells were seeded in 24-well plates at a density of 4 × 104 cells/well in 1 ml growth medium in a humidified atmosphere with 5% CO2 . After that, the various concentrations of chitosan/n-HA composite membrane extracts (25%, 50%, 75% and 100%) were added to wells for 24 h’s incubation. After 24 h’s incubation, 200 l of MTT solution (5 mg/ml) was added to corresponding wells and cultured at 37 ◦ C for another 4 h to allow the formation of formazan crystals. Finally, 250 l mixture was collected and transferred into a 96-well plate to record the absorbance at 570 nm using a Microplate reader (Bio-Rad, USA). The cell viability (%) was related to the control wells containing untreated cells with fresh cell culture medium and was calculated according to the following: cell viability (%) = absorption test/absorption control × 100%. All results were the mean values of three specimens.
3. Results and discussion 3.1. Preparation and characterization of n-HA A typical TEM photograph of n-HA crystals was presented in Fig. 1. From Fig. 1, we could find that the prepared n-HA was needlelike and the size is about 20 nm in vertical diameter and about 100 nm in horizontal diameter suitable for bone tissue engineering.
Fig. 1. TEM photograph of the nano-hydroxyapatite (n-HA).
3.2. Preparation and characterization of n-HA/chitosan cross-linking composite membrane 3.2.1. FTIR analysis The FTIR spectra of pure n-HA, blank chitosan membrane and nHA/chitosan cross-linking composite membrane was presented in Fig. 2. As the previous description of Fu et al. [30], the characteristic spectra of pure n-HA was corresponding to: –OH (3500 cm−1 ) and 1110 cm−1 was belong to the PO4 3− in n-HA. And the characteristic spectra of unmodified chitosan membrane was corresponding to: C–O–C (1071 cm−1 ) and –NH2 (1598 cm−1 and 1640 cm−1 ), while the band at 3430 cm−1 belongs to the stretching vibrations of the hydroxyl groups via hydrogen bonds. From Fig. 2, we could find that the amino stretching bands from blank chitosan membrane was shifted to the lower wavenumbers (1553 cm−1 and 1631 cm−1 ), indicating that the partial amino group of chitosan was crosslinking by genipin [19,20]. For the spectrum of n-HA/chitosan cross-linking composite membrane, we could find that the characteristic band of amino group from chitosan at 1631 cm−1 was disappeared while the amide I band at 1548 cm−1 became less intense, indicating that there was existence of possible electronic interaction and hydrogen bond between the PO4 3− of n-HA and NH3 + of chitosan [2,3]. On the other hand, the characteristic band of n-HA from n-HA/chitosan cross-linking composite membrane became less intense and shifted to the lower wavenumbers (from 1110 cm−1 to 1026 cm−1 ) as compared with that of pure n-HA, also implying that there was existence of possible electronic interaction between polymer and n-HA. Therefore, with the analysis of FTIR spectra, we speculated that there were presence of possible physical interaction (electronic interaction and hydrogen bond) rather than chemical reaction between n-HA and chitosan. 3.2.2. Crystallographic assay Fig. 3 shows the X-ray diffraction patterns of pure n-HA, blank chitosan membrane and n-HA/chitosan cross-linking composite membranes. According to the previous report [26], the semicrystalline chitosan exhibited a reflection peak at about 2 = 25◦ and a relatively weak reflection at 2 = 10◦ , which were assigned to two different crystal forms. As depicted in Fig. 3, the X-ray diffraction pattern of blank chitosan membrane showed three reflection peaks at about 10◦ , 18◦ and 25◦ , respectively. The new peak at about 2 = 18◦ might be induced by the resident of cross-link agent (genipin) in the membrane [31]. The typical crystalline peaks of n-HA at 2 = 25.8◦ , 32.2◦ , 33◦ , 34.1◦ , 35.5◦ and 40◦ , which is the similar to that of n-HA in natural bone. For the spectrum of nHA/chitosan cross-linking composite membrane, the specific peaks for chitosan at 2 = 10◦ , 18◦ and 25◦ were weaken, indicating that the introduction of n-HA into chitosan membrane could interfere the crystallinity of chitosan, yet resulting the distinction of XRD
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Fig. 2. FTIR spectra of pure HA, blank chitosan membrane and n-HA/chitosan cross-linking composite membrane (n-HA; 30 wt%).
spectra [1]. Additionally, the main specific peaks of n-HA were clearly observed in the composite membrane implying that n-HA was not be changed as other Ca–P phase during fabrication process [32]. 3.2.3. TGA analysis Thermal decomposition behavior of blank chitosan membrane and n-HA/chitosan cross-linking composite membranes was characterized by TG analysis. As presented in Fig. 4, it clearly observed that there was two-stages degradation behavior for the blank chitosan membrane. The weight loss at 104 ◦ C might be induced by the presence of bond water in the samples and weight loss at approximately 265 ◦ C was attributed to the degradation of chitosan [19]. For n-HA/chitosan cross-linking composite membranes, there was a new degradation peak at about 175 ◦ C, which might be attributed to the presence of water bond to the n-HA in the composite membrane [19]. Meanwhile, the n-HA content was greatly influenced on the degradation behavior of composite film. The temperature of degradation (Td ) of n-HA/chitosan cross-linking composite membrane was gradually increased from 265 ◦ C to 281 ◦ C as the n-HA content increasing from 0% to 15%, which might be explained by that there was presence of possible interaction (hydrogen bond and electronic interaction) between two components, yet resulting in the increase of Td . Li et al. [31] also demonstrated that blending n-HA with the chitosan could significantly elevate the degradation temperature of chitosan by the possible interaction between two components. Following the n-HA content increasing to 20%
Fig. 3. XRD patterns of blank chitosan film, pure n-HA and n-HA/chitosan crosslinking composite membrane (n-HA; 30 wt%).
and 30%, an interesting phenomenon was observed that the Td of n-HA/chitosan cross-linking composite membrane decreased dramatically from 281 ◦ C to 273 ◦ C, which might be attributed to the redundant n-HA in composite membrane could destroy integrity of membrane as well as interaction between components resulting in the decrease of Td [33]. The relative higher Td of n-HA/chitosan cross-linking composite membranes (S2, S3 and S4) compared with that of blank chitosan membrane implying that there are presence of possible interaction (hydrogen bond and electronic interaction) between chitosan and n-HA, which was in accordance with the prediction of FTIR. 3.2.4. Morphological analysis According to the SEM observation (Fig. 5), we could find that the morphology of upper surface and bottom surface of composite membrane showed the obvious discrepancy. The discrepancy of upper surface and bottom surface of composite membrane might have greatly effect on the properties of composite membrane as well as the application of membrane. As presented in Fig. 5(A-1, A-2), the both surface of blank chitosan membrane were smooth. With the n-HA content increased from 5% to 30% (Fig. 5(B–F)), the roughness of both surface of composite membrane increased obviously due to the separation of n-HA crystal dispersed throughout composite membrane [1,32]. However, bottom surface of n-HA/chitosan cross-linking composite membrane was more roughness than upper surface of composite membrane, which might be attributed to the precipitation of n-HA during the membrane fabrication process [33]. Meanwhile, it also revealed
Fig. 4. TG curves of blank chitosan membrane and n-HA/chitosan cross-linking composite membranes.
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Fig. 5. Scanning electron microscopy (SEM) micrographs: (A-1) upper surfaces of S1; (B-1) upper surfaces of S2; (C-1) upper surfaces of S3; (D-1) upper surfaces of S4; (E-1) upper surfaces of S5; (F-1) upper surfaces of S6. (A-2) bottom surfaces of S1; (B-2) bottom surfaces of S2; (C-2) bottom surfaces of S3; (D-2) bottom surfaces of S4; (E-2) bottom surfaces of S5; (F-2) bottom surfaces of S6.
that the bottom surface of composite membrane was rift as the n-HA content was greater than 15%, which could be see clearly by 10,000× magnification in Fig. 5(D–F). However, this discontinuous structure of composite membrane was disadvantage for the further in vivo application. Therefore, in order to obtain the homogenous n-HA/chitosan cross-linking composite membrane, the n-HA content in the composite membrane should be carefully controlled within 15 wt%. Furthermore, the proceeding method of n-HA/chitosan cross-linking composite membrane was also needed further improvement.
n-HA content increased to 20% or even up to 30%, the serious aggregation or agglomeration of n-HA particles was observed in the composite membrane, yet resulting in the discontinuous structure of membrane (Fig. 5). Therefore, the tensile strength of composite membrane decreased dramatically from 73.50 ± 7.67 MPa to 31.73 ± 6.56 MPa as the n-HA content was greater than 15% [34]. Above on the information, in order to obtain a homogeneous composite membrane with well mechanical property, the n-HA content in the composite membrane should be carefully controlled. 3.3. Water adsorption test
3.2.5. Mechanical properties Mechanical property is an important property for bioceramic/polymer hybrid membranes which potential used in tissue engineering. In order to investigate the effect of n-HA content on the mechanical property of the n-HA/chitosan cross-linking composite membranes, tensile test were performed. The tensile strength, elongation rate and Young’s modulus of the composite membranes were obtained and listed in Table 2. According to Fig. 6, it revealed that the tensile strength of composite membrane showed great dependence on the n-HA content. The tensile strength increased from 36.45 ± 5.34 MPa to 73.50 ± 7.67 MPa as the n-HA content increasing from 0% to 10%. Following the n-HA content increased from 15% to 30%, the tensile strength decreased dramatically from 73.50 ± 7.67 MPa to 31.73 ± 6.56 MPa. This interesting phenomenon might be explained by that n-HA particles could disperse homogeneously in the chitosan–genipin solution and did not separated from the membrane during the fabrication process as the n-HA content was lower than 15%. On the other hand, due to presence of possible interaction (hydrogen bonds and electronic interaction) between two components as predicted by FTIR and TGA measurement, the tensile strength of composite membrane could also be improved. Conversely, following the
The ability for a membrane to preserve water is one of the most important aspects in tissue engineering. As presented in Fig. 7, the
Fig. 6. Stress-strain of n-HA/chitosan cross-linking composite membrane.
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Table 2 Mechanical properties of blank chitosan membrane and n-HA/chitosan cross-linking composite membrane (Data as mean± standard error of mean; n = 5). Samples
Tensile strength (MPa)
S-1 S-2 S-3 S-4 S-5 S-6
36.45 48.51 73.50 68.98 58.94 31.73
± ± ± ± ± ±
5.34 7.43 7.67 5.45 4.56 6.56
water adsorption of composite membrane decreased from 1800% to 830% as n-HA content increasing from 0% to 30%. As the previous report, water is absorbed into the membranes by two processes: water binding to the materials itself and water being retained in pore space. For the developed n-HA/chitosan cross-linking composite membrane, due to absence any pore structure of membrane, water adsorption mainly induced by the water bond to the chitosan itself. As well known to us, chitosan containing primary amine (–NH2) and hydroxyl group (–OH) can not only increase its affinity to water but also form hydrogen bonds with water [18,25]. But due to the poor hydrophilicity of n-HA, the introduction of n-HA to chitosan membrane could obviously decrease the hydrophilicity of composite membrane, thus resulting in the decrease of water adsorption. Moreover, the possible interaction between chitosan and n-HA predicated by TGA and FTIR could also decrease the hydrogen bond between water and chitosan, also resulting in the decrease of water adsorption.
3.4. In vitro cytotoxicity test Previous studies have demonstrated that the chitosan, genipin and n-HA were non-cytotoxicity against HK293 cells [19]. However, the cytotoxicity of n-HA/chitosan cross-linking composite membrane has yet not been evaluated. In this paper, L929 cells were used to evaluate the intrinsic cytotoxicity of n-HA/chitosan cross-linking composite membrane by an indirect method. The composite membrane with 30% n-HA was selected to evaluate the cytotoxicity of n-HA/chitosan cross-linking composite membrane, due to the highest n-HA content in the series membrane. Fig. 8 depicts the cell viability as function with the concentration of extract fluids assessed by MTT test. According to Fig. 8, we could find that all extraction of membranes were non cytotoxicity against the L929 cells after 24 h’s incubation, indicating that the developed n-HA/chitosan cross-linking composite membranes was non-cytotoxicity against L929 in vitro might be suitable for further in vivo application.
Elongation at break (%) 3.67 4.06 3.81 4.64 4.61 2.80
± ± ± ± ± ±
0.67 0.48 1.62 0.33 1.92 0.88
Young’s modulus (Mpa) 2345.56 2514.76 3597.68 3796.13 3178.81 2277.21
± ± ± ± ± ±
21.23 16.47 87.93 102.25 96.63 153.46
Fig. 8. In vitro cytotoxicity for blank chitosan membrane and n-HA/chitosan crosslinking composite membrane (30 wt%). L929 cells were incubated with different concentrations of extracts obtained from blank chitosan membrane, n-HA/chitosan cross-linking composite membrane.
4. Conclusion In this paper, a series of cross-linking chitosan membrane made from various n-HA contents (0, 5, 10, 15, 20 and 30 wt%) were developed by a casting/solvent evaporation method. According to the analysis of FTIR and TGA measurement, there is presence of the possible physical interactions (hydrogen bond and electric interaction) between two components. The n-HA content was greatly effect on the property of composite membrane. As the n-HA content was greater than 10%, the serious separation of n-HA from the composite membrane was clearly observed, yet resulting in the discontinuous structure of membrane with SEM observation. By changing the n-HA content in composite membrane, the various water adsorption ranging from 830% to 1800% and tensile strength ranging from 30 MPa to 73 MPa could be gained. Meanwhile, in vitro cytotoxicity test revealed that the prepared n-HA/chitosan crosslinking composite membranes were non-cytotoxicity against L929 cells after 24 h’s incubation. Therefore, we suggested that the developed n-HA/chitosan cross-linking composite membranes with well cytocompatibility, water adsorption and suitable tensile strength might can be served as the vehicle for the bone tissue engineering application. References
Fig. 7. Water adsorption of n-HA/chitosan cross-linking composite membrane.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Preparation and characterization of nano-hydroxyapatite/chitosan cross-linking composite membrane intended for tissue engineering XingYi Li a,∗ , KaiHui Nan a , Shuai Shi b , Hao Chen a,∗ a b
Institute of Biomedical Engineering, School of Ophthalmology & Optometry and Eye hospital, Wenzhou Medical College, 270 Xueyuan Road, Wenzhou 325027, China State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, No. 1, Keyuan 4th Road, Chengdu 610041, China
a r t i c l e
i n f o
Article history: Received 9 August 2011 Received in revised form 5 September 2011 Accepted 24 September 2011 Available online 1 October 2011 Keywords: Chitosan Nano-hydroxyapatite In vitro cytotoxicity Composite membrane Cross-linking
a b s t r a c t In this paper, a series of nano-hydroxyapatite(n-HA)/chitosan cross-linking composite membranes (nHA; 0, 5, 10, 15, 20 and 30 wt%) were successfully developed by a simple casting/solvent evaporation method. n-HA with size about 20 nm in vertical diameter and about 100 nm in horizontal diameter was successfully synthesized by a hydro-thermal precipitation method, and then dispersed into chitosan/genipin solution with the aid of continuous ultrasound to develop n-HA/chitosan cross-linking composite membranes. The detailed characterizations including Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water adsorption and tensile test were performed. With the analysis of FTIR spectra and TGA spectra, it suggested that there was existence of possible interactions between polymer and n-HA. Meanwhile, the n-HA content was greatly effected on the morphology as well as the tensile property of composite membrane. In vitro cytotoxicity test suggested that the developed n-HA/chitosan cross-linking composite membrane was non-cytotoxicity against L929 cells after 24 h’s incubation might be suitable for further in vivo application. © 2011 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. In past several decades, polymer blend membrane/film has been widely used for tissue engineering and other biomedical applications [1,2]. As well known to us, an ideal membrane for tissue engineering is strongly desired to have good mechanical property, swelling behavior, biocompatibility, bioactivity, and so on [3]. Nowadays, some non-biodegradable membranes, such as expanded polytetrafluoroethylene (e-PTFE) and so on have been clinically employed as barrier membranes for guided bone regeneration [4,5]. However, as the non-biodegradable/absorbable matrix, the implant membrane must be removed out from the body by a second surgical procedure after the healing of bone defect, which will result in the risk of tissue morbidity. Therefore, more and more attention have been oriented to the biodegradable/absorbable membranes made from the
∗ Corresponding authors at: School of Ophthalmology & Optometry and Eye hospital, Wenzhou Medical College, 270 Xueyuan Road, China. Tel.: +86 577 88833806; fax: +86 577 88833806. E-mail addresses: lixingyi [email protected] (X. Li), [email protected] (H. Chen). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.09.021
natural/synthetic polymers such as polylactic acid, chitosan, alginate, and collagen [6–8]. Among these biodegradable/absorbable membranes, chitosan membrane/film has gained considerable attention because of its favorable properties such as low-toxicity, good biocompatibility, biodegradability, mucoadhesive, and easy fabrication [9]. As the cationic polysaccharide in nature, chitosan is composed of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, only dissolve in some acidic solution as pH value was below 6 [10,11]. In past three decades, it has been studied intensively for its applications in pharmaceutical science, cosmetics, biomedical fields, agriculture, and food industries as well as in sewage treatment, paper industry [12–15]. Recently, chitosan and its derivatives have been successfully fabricated into films/membranes for various medical applications such as tissue engineering, wound healing and so on [12,16]. However, some shortcoming made chitosan membrane/film unwelcome in medical application due to its high sensitivity to water as well as only dissolve in acidic solution during the fabrication process. In order to exploit the unique properties of this versatile cationic polysaccharide, several attempts are being made to cross-linking chitosan membrane/film to modulate its hydrophobic–hydrophilic balance and mechanical property, realizing its full potential applications [17,18]. Genipin, as a water soluble bi-functional cross-linking reagent, has been widely used to cross-linking chitosan matrix, proteins and amine in general [19]. Meanwhile, the resulting crosslinked complexes are
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non-cytotoxicity for the animal and human cells as so far examined. The safety and the beneficial actions of genipin has been used a number of research projects in the areas of the therapies of diabetes, periodontitis, cataract, hepatic dysfunction, as well as in wound repair and nerve regeneration [19,20]. Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HA) has been widely used in tissue engineering fields due to its shape was needle-like and its structure and composition similar to minerals of natural bones [21–23]. It not only plays a primary role in improving the mechanical properties of the nanocomposites, but also provides a favorable environment for osteoconduction, protein adhesion, and osteoblast proliferation. Biopolymer/n-HA composites membranes/films have been prepared by many methods like blending, biomimetic process using simulated body fluid (SBF), in situ precipitation and electrochemical deposition [24,25]. Among these methods, the blending is a simple method to develop the n-HA/chitosan cross-linking composite film, that is to say, n-HA/chitosan cross-linking composite membrane could be obtained by simple dispersing n-HA into chitosan–genipin solution. The cross-linking degree of membranes as well as the mechanical property could be modulated by changing the genipin concentration in the formulation, and the unreactive genipin could be extracted by immersion of membrane in phosphate buffered solution (PBS) overnight. Furthermore, the application of genipin to cross-link the composite membrane could significantly improve the hydrophobic property of the composite membrane. The obtained series of n-HA loaded chitosan composite membranes were carefully characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water adsorption and tensile test before its further in vivo application.
2. Materials and methods 2.1. Materials Chitosan (92% degree of deacetylation (DD)) with molecular weight about 200 kDa was supplied by Sigma–Aldrich (USA). Genipin was purchased from Shanghai Y-S Biotechnology Co. LTD (China). Ca(NO3 )·4H2 O and (NH4 )2 HPO4 ·3H2 O were obtained from WenZhou Chemical Reagents Co. LTD (China). All other chemicals used in this paper were analytical grade. Ultrapure water from Milli-Q water system was used to prepare the aqueous solutions.
2.2. Preparation and characterization of nano-hydroxyapatite (n-HA) Pure nano-hydroxyapatite (n-HA) powder was synthesized by a hydro-thermal precipitation method as previous report with little modification [16]. Ca(NO3 )·4H2 O and (NH4 )2 HPO4 ·3H2 O were used as starting materials, and the reaction was performed according to the following reaction: Ca(NO3 )·4H2 O + (NH4 )2 HPO4 ·3H2 O → Ca10 (PO4 )6 (OH)2 The pH value of reaction system was adjusted to 10 by addition of ammonium hydroxide (NH3 ·H2 O) solution. The obtained raw HA precipitation was first treated at 80 ◦ C under normal atmospheric pressure for 2 h. And then the precipitation was centrifuged and fully washed to neutrality by distilled water and anhydrous alcohol. Finally, the obtained n-HA slurry was sintered at 1000 ◦ C for 2 h, and then ground into powder. The morphological characterization of nHA was observed with a transmission electron microscopy (TEM) (H-6009IV, Hitachi, Japan).
Table 1 n-HA/chitosan cross-linking composite membrane made from various n-HA contents. Samples
Chitosan concentration (mg/ml)
n-HA content (%)
Genipin concentration (mM)
S-1 S-2 S-3 S-4 S-5 S-6
20 20 20 20 20 20
0 5 10 15 20 30
0.5 0.5 0.5 0.5 0.5 0.5
2.3. Preparation and characterization of n-HA/chitosan cross-linking composite membrane 2.3.1. Preparation of n-HA/chitosan cross-linking composite membrane The casting/solvent evaporation technology was employed to develop the n-HA/chitosan cross-linking composite membranes [25,26]. Initially, chitosan (4 g) was dissolved in 1% acetic acid solution (200 ml) under magnetic stirring for 48 h at room temperature. After that, genipin was added to the above solution with the final concentration at 0.5 mM. Subsequently, the obtained n-HA powder regardless of various weight ratios of n-HA/chitosan, was added to the above chitosan–genipin solution with the continuous ultrasound to obtain the homogeneous n-HA/chitosan–genipin suspension. Finally, 20 ml n-HA/chitosan–genipin suspension was poured into a glass Petri dish (d = 6 cm) and dried at 37 ◦ C for 2 days to obtain the n-HA/chitosan cross-linking composite membranes, as shown in Table 1. In order to remove the resident acetic acid and unreactive genipin, the obtained n-HA/chitosan cross-linking composite membranes were immersed into phosphate buffered saline (PBS) solution for about 6 h, and then washed with distilled water twice to neutral. All membranes were re-dried at 37 ◦ C for 2 days before the further characterization and application. 2.3.2. Fourier transform infrared spectroscopy (FTIR) measurement FTIR (KBr pellet) spectra were performed at room temperature using a NICOLET 200SXV Infrared Spectrophotometer (USA). The characteristic absorption bands of the composite membranes were detected at wavenumbers ranging from 500 to 4000 cm−1 . 2.3.3. Crystallographic assay X-ray diffraction spectrometry was obtained by using X-ray Diffractometer (DX-2000, DanDong Fangyuan Instrument Company, China) using CuKa radiation. The relative intensities were recorded within the range of 10–60◦ (2) at a scanning rate of 4◦ min−1 . 2.3.4. Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed by a thermogravimetric analyzer (TA 2910, DuPont, USA) under a steady flow of nitrogen atmosphere at a heating rate of 10 ◦ C/min in the range of 20–600 ◦ C. 2.3.5. Morphological analysis The morphological characterization of composite membrane was performed by scanning electron microscopy (JSM-5900LV, JEOL, Japan). Composite membrane samples were placed at cabinet drier for 24 h before the observation. The upper and bottom surface of composite membrane was observed respectively.
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2.3.6. Tensile test Rectangular specimens of the n-HA/chitosan crosslinking composite membranes with dimensions of 40 mm × 5 mm × 0.06–0.10 mm were tested by a universal mechanical testing instrument (Instron-5567, Instron Corp., USA) at room temperature and relative humidity of 50%. The tensile strength and elongation were evaluated at a displacement rate of 20 mm/min with 20-mm gauge length. All results were mean values of five specimens.
2.4. Water adsorption studies Water adsorption of n-HA/chitosan cross-linking composite membranes was detected by weighing the membranes pieces before and after placing in 20 ml distilled water solution. Each membrane was divided in portions of 1 cm2 (1 cm × 1 cm), weighed and placed in distilled water solution for periodical study (5, 10, 20, 40, 60 and 90 min) as described by Rodrigues et al. [27]. At specific time intervals, the membranes were taken from the medium and weighed after removal of the surplus surface water using filter paper. The water absorption were calculated by following: Water adsorption (%) =
W90 − W0 × 100 W0
(1)
where W90 is the weight of wet membrane at 90 min and W0 is the original membrane weight at dry state, respectively. Here, the water absorption of composite membrane after immersed in distilled water solution for 90 min was defined as equilibriumswelling ratio. This experiment was performed in triplicate.
2.5. In vitro cytotoxicity test According to the previous studies, MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) test was used to evaluate in vitro cytotoxicity of n-HA/chitosan cross-linking composite membranes [28,29]. The mouse fibroblastic-like cells (L929 cell) were cultured with DMEM medium at 37 ◦ C and 5% CO2 . Due to the insoluble of composite membrane in water solution, the leachables of polymeric component from the n-HA/chitosan cross-linking composite membrane was employed to evaluate the cytotoxicity of composite membrane. Initially, cells were seeded in 24-well plates at a density of 4 × 104 cells/well in 1 ml growth medium in a humidified atmosphere with 5% CO2 . After that, the various concentrations of chitosan/n-HA composite membrane extracts (25%, 50%, 75% and 100%) were added to wells for 24 h’s incubation. After 24 h’s incubation, 200 l of MTT solution (5 mg/ml) was added to corresponding wells and cultured at 37 ◦ C for another 4 h to allow the formation of formazan crystals. Finally, 250 l mixture was collected and transferred into a 96-well plate to record the absorbance at 570 nm using a Microplate reader (Bio-Rad, USA). The cell viability (%) was related to the control wells containing untreated cells with fresh cell culture medium and was calculated according to the following: cell viability (%) = absorption test/absorption control × 100%. All results were the mean values of three specimens.
3. Results and discussion 3.1. Preparation and characterization of n-HA A typical TEM photograph of n-HA crystals was presented in Fig. 1. From Fig. 1, we could find that the prepared n-HA was needlelike and the size is about 20 nm in vertical diameter and about 100 nm in horizontal diameter suitable for bone tissue engineering.
Fig. 1. TEM photograph of the nano-hydroxyapatite (n-HA).
3.2. Preparation and characterization of n-HA/chitosan cross-linking composite membrane 3.2.1. FTIR analysis The FTIR spectra of pure n-HA, blank chitosan membrane and nHA/chitosan cross-linking composite membrane was presented in Fig. 2. As the previous description of Fu et al. [30], the characteristic spectra of pure n-HA was corresponding to: –OH (3500 cm−1 ) and 1110 cm−1 was belong to the PO4 3− in n-HA. And the characteristic spectra of unmodified chitosan membrane was corresponding to: C–O–C (1071 cm−1 ) and –NH2 (1598 cm−1 and 1640 cm−1 ), while the band at 3430 cm−1 belongs to the stretching vibrations of the hydroxyl groups via hydrogen bonds. From Fig. 2, we could find that the amino stretching bands from blank chitosan membrane was shifted to the lower wavenumbers (1553 cm−1 and 1631 cm−1 ), indicating that the partial amino group of chitosan was crosslinking by genipin [19,20]. For the spectrum of n-HA/chitosan cross-linking composite membrane, we could find that the characteristic band of amino group from chitosan at 1631 cm−1 was disappeared while the amide I band at 1548 cm−1 became less intense, indicating that there was existence of possible electronic interaction and hydrogen bond between the PO4 3− of n-HA and NH3 + of chitosan [2,3]. On the other hand, the characteristic band of n-HA from n-HA/chitosan cross-linking composite membrane became less intense and shifted to the lower wavenumbers (from 1110 cm−1 to 1026 cm−1 ) as compared with that of pure n-HA, also implying that there was existence of possible electronic interaction between polymer and n-HA. Therefore, with the analysis of FTIR spectra, we speculated that there were presence of possible physical interaction (electronic interaction and hydrogen bond) rather than chemical reaction between n-HA and chitosan. 3.2.2. Crystallographic assay Fig. 3 shows the X-ray diffraction patterns of pure n-HA, blank chitosan membrane and n-HA/chitosan cross-linking composite membranes. According to the previous report [26], the semicrystalline chitosan exhibited a reflection peak at about 2 = 25◦ and a relatively weak reflection at 2 = 10◦ , which were assigned to two different crystal forms. As depicted in Fig. 3, the X-ray diffraction pattern of blank chitosan membrane showed three reflection peaks at about 10◦ , 18◦ and 25◦ , respectively. The new peak at about 2 = 18◦ might be induced by the resident of cross-link agent (genipin) in the membrane [31]. The typical crystalline peaks of n-HA at 2 = 25.8◦ , 32.2◦ , 33◦ , 34.1◦ , 35.5◦ and 40◦ , which is the similar to that of n-HA in natural bone. For the spectrum of nHA/chitosan cross-linking composite membrane, the specific peaks for chitosan at 2 = 10◦ , 18◦ and 25◦ were weaken, indicating that the introduction of n-HA into chitosan membrane could interfere the crystallinity of chitosan, yet resulting the distinction of XRD
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Fig. 2. FTIR spectra of pure HA, blank chitosan membrane and n-HA/chitosan cross-linking composite membrane (n-HA; 30 wt%).
spectra [1]. Additionally, the main specific peaks of n-HA were clearly observed in the composite membrane implying that n-HA was not be changed as other Ca–P phase during fabrication process [32]. 3.2.3. TGA analysis Thermal decomposition behavior of blank chitosan membrane and n-HA/chitosan cross-linking composite membranes was characterized by TG analysis. As presented in Fig. 4, it clearly observed that there was two-stages degradation behavior for the blank chitosan membrane. The weight loss at 104 ◦ C might be induced by the presence of bond water in the samples and weight loss at approximately 265 ◦ C was attributed to the degradation of chitosan [19]. For n-HA/chitosan cross-linking composite membranes, there was a new degradation peak at about 175 ◦ C, which might be attributed to the presence of water bond to the n-HA in the composite membrane [19]. Meanwhile, the n-HA content was greatly influenced on the degradation behavior of composite film. The temperature of degradation (Td ) of n-HA/chitosan cross-linking composite membrane was gradually increased from 265 ◦ C to 281 ◦ C as the n-HA content increasing from 0% to 15%, which might be explained by that there was presence of possible interaction (hydrogen bond and electronic interaction) between two components, yet resulting in the increase of Td . Li et al. [31] also demonstrated that blending n-HA with the chitosan could significantly elevate the degradation temperature of chitosan by the possible interaction between two components. Following the n-HA content increasing to 20%
Fig. 3. XRD patterns of blank chitosan film, pure n-HA and n-HA/chitosan crosslinking composite membrane (n-HA; 30 wt%).
and 30%, an interesting phenomenon was observed that the Td of n-HA/chitosan cross-linking composite membrane decreased dramatically from 281 ◦ C to 273 ◦ C, which might be attributed to the redundant n-HA in composite membrane could destroy integrity of membrane as well as interaction between components resulting in the decrease of Td [33]. The relative higher Td of n-HA/chitosan cross-linking composite membranes (S2, S3 and S4) compared with that of blank chitosan membrane implying that there are presence of possible interaction (hydrogen bond and electronic interaction) between chitosan and n-HA, which was in accordance with the prediction of FTIR. 3.2.4. Morphological analysis According to the SEM observation (Fig. 5), we could find that the morphology of upper surface and bottom surface of composite membrane showed the obvious discrepancy. The discrepancy of upper surface and bottom surface of composite membrane might have greatly effect on the properties of composite membrane as well as the application of membrane. As presented in Fig. 5(A-1, A-2), the both surface of blank chitosan membrane were smooth. With the n-HA content increased from 5% to 30% (Fig. 5(B–F)), the roughness of both surface of composite membrane increased obviously due to the separation of n-HA crystal dispersed throughout composite membrane [1,32]. However, bottom surface of n-HA/chitosan cross-linking composite membrane was more roughness than upper surface of composite membrane, which might be attributed to the precipitation of n-HA during the membrane fabrication process [33]. Meanwhile, it also revealed
Fig. 4. TG curves of blank chitosan membrane and n-HA/chitosan cross-linking composite membranes.
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Fig. 5. Scanning electron microscopy (SEM) micrographs: (A-1) upper surfaces of S1; (B-1) upper surfaces of S2; (C-1) upper surfaces of S3; (D-1) upper surfaces of S4; (E-1) upper surfaces of S5; (F-1) upper surfaces of S6. (A-2) bottom surfaces of S1; (B-2) bottom surfaces of S2; (C-2) bottom surfaces of S3; (D-2) bottom surfaces of S4; (E-2) bottom surfaces of S5; (F-2) bottom surfaces of S6.
that the bottom surface of composite membrane was rift as the n-HA content was greater than 15%, which could be see clearly by 10,000× magnification in Fig. 5(D–F). However, this discontinuous structure of composite membrane was disadvantage for the further in vivo application. Therefore, in order to obtain the homogenous n-HA/chitosan cross-linking composite membrane, the n-HA content in the composite membrane should be carefully controlled within 15 wt%. Furthermore, the proceeding method of n-HA/chitosan cross-linking composite membrane was also needed further improvement.
n-HA content increased to 20% or even up to 30%, the serious aggregation or agglomeration of n-HA particles was observed in the composite membrane, yet resulting in the discontinuous structure of membrane (Fig. 5). Therefore, the tensile strength of composite membrane decreased dramatically from 73.50 ± 7.67 MPa to 31.73 ± 6.56 MPa as the n-HA content was greater than 15% [34]. Above on the information, in order to obtain a homogeneous composite membrane with well mechanical property, the n-HA content in the composite membrane should be carefully controlled. 3.3. Water adsorption test
3.2.5. Mechanical properties Mechanical property is an important property for bioceramic/polymer hybrid membranes which potential used in tissue engineering. In order to investigate the effect of n-HA content on the mechanical property of the n-HA/chitosan cross-linking composite membranes, tensile test were performed. The tensile strength, elongation rate and Young’s modulus of the composite membranes were obtained and listed in Table 2. According to Fig. 6, it revealed that the tensile strength of composite membrane showed great dependence on the n-HA content. The tensile strength increased from 36.45 ± 5.34 MPa to 73.50 ± 7.67 MPa as the n-HA content increasing from 0% to 10%. Following the n-HA content increased from 15% to 30%, the tensile strength decreased dramatically from 73.50 ± 7.67 MPa to 31.73 ± 6.56 MPa. This interesting phenomenon might be explained by that n-HA particles could disperse homogeneously in the chitosan–genipin solution and did not separated from the membrane during the fabrication process as the n-HA content was lower than 15%. On the other hand, due to presence of possible interaction (hydrogen bonds and electronic interaction) between two components as predicted by FTIR and TGA measurement, the tensile strength of composite membrane could also be improved. Conversely, following the
The ability for a membrane to preserve water is one of the most important aspects in tissue engineering. As presented in Fig. 7, the
Fig. 6. Stress-strain of n-HA/chitosan cross-linking composite membrane.
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Table 2 Mechanical properties of blank chitosan membrane and n-HA/chitosan cross-linking composite membrane (Data as mean± standard error of mean; n = 5). Samples
Tensile strength (MPa)
S-1 S-2 S-3 S-4 S-5 S-6
36.45 48.51 73.50 68.98 58.94 31.73
± ± ± ± ± ±
5.34 7.43 7.67 5.45 4.56 6.56
water adsorption of composite membrane decreased from 1800% to 830% as n-HA content increasing from 0% to 30%. As the previous report, water is absorbed into the membranes by two processes: water binding to the materials itself and water being retained in pore space. For the developed n-HA/chitosan cross-linking composite membrane, due to absence any pore structure of membrane, water adsorption mainly induced by the water bond to the chitosan itself. As well known to us, chitosan containing primary amine (–NH2) and hydroxyl group (–OH) can not only increase its affinity to water but also form hydrogen bonds with water [18,25]. But due to the poor hydrophilicity of n-HA, the introduction of n-HA to chitosan membrane could obviously decrease the hydrophilicity of composite membrane, thus resulting in the decrease of water adsorption. Moreover, the possible interaction between chitosan and n-HA predicated by TGA and FTIR could also decrease the hydrogen bond between water and chitosan, also resulting in the decrease of water adsorption.
3.4. In vitro cytotoxicity test Previous studies have demonstrated that the chitosan, genipin and n-HA were non-cytotoxicity against HK293 cells [19]. However, the cytotoxicity of n-HA/chitosan cross-linking composite membrane has yet not been evaluated. In this paper, L929 cells were used to evaluate the intrinsic cytotoxicity of n-HA/chitosan cross-linking composite membrane by an indirect method. The composite membrane with 30% n-HA was selected to evaluate the cytotoxicity of n-HA/chitosan cross-linking composite membrane, due to the highest n-HA content in the series membrane. Fig. 8 depicts the cell viability as function with the concentration of extract fluids assessed by MTT test. According to Fig. 8, we could find that all extraction of membranes were non cytotoxicity against the L929 cells after 24 h’s incubation, indicating that the developed n-HA/chitosan cross-linking composite membranes was non-cytotoxicity against L929 in vitro might be suitable for further in vivo application.
Elongation at break (%) 3.67 4.06 3.81 4.64 4.61 2.80
± ± ± ± ± ±
0.67 0.48 1.62 0.33 1.92 0.88
Young’s modulus (Mpa) 2345.56 2514.76 3597.68 3796.13 3178.81 2277.21
± ± ± ± ± ±
21.23 16.47 87.93 102.25 96.63 153.46
Fig. 8. In vitro cytotoxicity for blank chitosan membrane and n-HA/chitosan crosslinking composite membrane (30 wt%). L929 cells were incubated with different concentrations of extracts obtained from blank chitosan membrane, n-HA/chitosan cross-linking composite membrane.
4. Conclusion In this paper, a series of cross-linking chitosan membrane made from various n-HA contents (0, 5, 10, 15, 20 and 30 wt%) were developed by a casting/solvent evaporation method. According to the analysis of FTIR and TGA measurement, there is presence of the possible physical interactions (hydrogen bond and electric interaction) between two components. The n-HA content was greatly effect on the property of composite membrane. As the n-HA content was greater than 10%, the serious separation of n-HA from the composite membrane was clearly observed, yet resulting in the discontinuous structure of membrane with SEM observation. By changing the n-HA content in composite membrane, the various water adsorption ranging from 830% to 1800% and tensile strength ranging from 30 MPa to 73 MPa could be gained. Meanwhile, in vitro cytotoxicity test revealed that the prepared n-HA/chitosan crosslinking composite membranes were non-cytotoxicity against L929 cells after 24 h’s incubation. Therefore, we suggested that the developed n-HA/chitosan cross-linking composite membranes with well cytocompatibility, water adsorption and suitable tensile strength might can be served as the vehicle for the bone tissue engineering application. References
Fig. 7. Water adsorption of n-HA/chitosan cross-linking composite membrane.
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