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Preparation and biocompatibility of chitosan microcarriers as biomaterial
Biochemical Engineering Journal 27 (2006) 269–274
Preparation and biocompatibility of chitosan microcarriers as biomaterial Xi-Guang Chen a,∗ , Cheng-Sheng Liu a , Chen-Guang Liu a , Xiang-Hong Meng a , Chong M. Lee b , Hyun-Jin Park c b
a The College of Marine Life Science, Ocean University of China, 5# Yushan Road Qingdao, Qingdao 266003, PR China The FSN Research Center, Department of Nutrition and Food Sciences, University of Rhode Island, Kingston RI 02881, USA c The Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea
Received 20 October 2004; received in revised form 4 July 2005; accepted 3 August 2005
Abstract Chitosan microcarriers (100–200 m) were prepared by the methods of emulsification and ethanol coagulant. It has smooth surface and was stable in phosphate buffer solution (PBS) of at pH 7.2 in the treatment of temperature 120 ◦ C and pressure 150 kPa. The chitosan microcarriers showed molecular affinity to the bovine serum proteins at pH 7.2. The adsorptive capacity of the microcarriers to the serum albumin was 6.8 mg protein/g chitosan bead. The chitosan microcarriers were found to have good biocompatibility and no cytotoxicity to both human and mouse fibroblasts in tissue cell culture. The fibroblasts well adhered on the smooth surface of the chitosan microcarriers and grew in high cell density. The results suggest a good potential of the chitosan microcarriers as a wound-healing biomaterial. © 2005 Elsevier B.V. All rights reserved. Keywords: Affinity; Microcarrier; Tissue cell culture; Adsorption
1. Introduction Chitosan (-1,4-linked-2-amino-2-deoxy-d-glucopyranose), made from deacetylated chitin (-1,4-linked-2acetamido-2-deoxy-d-glucose) is a polysaccharide of considerable promise in the field of biomedical research. Their biodegradability, biocompatibility, and nontoxicity allow widespread applications in wound healing [1]. The ability of chitosan and chitin to form gels, films, fibers, and sponges demonstrate their inherent versatility [2–4]. Chitin beads are potentially useful as a wound dressing material [1]. Chitin-based wound dressing materials promote and accelerate wound healing. Chitin may possess a tissue cell growth function and may action as a favorable scaffold for cell attachment and proliferation. This promotes rapid dermal regeneration allow accelerated wound healing [5]. The formation of chitin beads has been extensively investigated. Methods to prepare chitin beads include emulsifi∗
Corresponding author. Tel.: +86 532 82032586; fax: +86 532 82032586. E-mail address: [email protected] (X.-G. Chen).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.08.021
cation, suspension and homogenization, freeze-drying and hammer milling [6–11]. The water insolubility of chitin is a limitation for the preparation process of the beads, some special solvents or compounds, such as hexafluoroisopropanol, hexafluoroacetone, chloroalcohols, need to be added into the solvent in order to make chitin solutions [2,12]. Chitosan is soluble in the diluted acetic acid solution, and can be made into microcarriers. Chitosan microcarriers were found to have more utility in drug carrier and delivery systems [13]. The preparation of chitosan microcarriers involves the crushing and precipitation of chitosan solution droplets in an anionic coagulant such as alginate, tripolyphosphate [14]. The emulsion process had been employed for the preparation of chitosan beads with smooth surface and uniform size distribution [15]. The chitosan microcarriers with smoothsurface are thought to be good condition for cells adherence and growth on it. The characteristics and biocompatibility of the chitosan microcarriers are necessary to be described. In this paper, a smooth-surface chitosan microcarriers were prepared by emulsion and cross-linking method. The characteristics, bio-affinity to bovine serum albumin, and
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X.-G. Chen et al. / Biochemical Engineering Journal 27 (2006) 269–274
cytotoxicity of the chitosan microcarriers were investigated. The cell growth status on the chitosan microcarriers in vitro was described.
the chitosan microcarriers were filtered and blotted dry on filter paper (Xinhua 1# Filter paper). 2.4. Affinity experiment
2. Materials and methods 2.1. Materials Chitosan (MW 480,000 Da, DD 90%) was made from crab-shell and obtained from Biochemical Medicine Plant of Qingdao (Qingdao, China). Bovine serum albumin (BSA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (Sigma Co., St. Louis, MO, USA). 2.2. Chitosan bead preparation The preparation of chitosan microcarriers were followed a patented procedure [16]. Chitosan powder was dissolved in 1% acetic acid to give a 2.5% chitosan solution. The solution was filtered through glass wool. Filtered chitosan solution (50 ml), tween-80 (2.5 ml) and toluene (150 ml) were added into a 500 ml flask. The mixture solution were stirred 30 min with an electromagnetic bar to form an emulsion. Formalin (5 ml) and glutaraldehyde (1 ml) were added dropwise into the emulsion and were keeping in stir for 1 h. The mixture was filtered through 200-mesh nylon screen to separate the microcarriers, and the microcarriers were suspended in1000 ml distilled water (containing 0.05 g NaH4 B in water) for 12 h. The microcarriers were filtered and rinsed in distilled water, then successively dewatered in 30, 50, 80, 95 and 100% ethanol and ether, respectively. 2.3. Characterization of chitosan microcarriers Scanning electron micrographs (SEM) were obtained with a Stereoscan 250 Mk3 (Cambridge, UK). Chitosan microcarriers were gold-coated in a JEOL JFC-1100 ion-sputter. The IR spectrum of chitosan and chitosan microcarriers were recorded on an FT/IR-430 Fourier Transform Infrared Spectrometer (Jasco Co., Tokyo, Japan) at room temperature based on the method of Shigemasa [17]. The samples were 2 mg in the 100 mg KBr to be made pellet [17]. Thermal stability of chitosan microcarriers was assessed by checking the numbers of the intact microcarrier beads after heating at 120 ◦ C under pressure of 150 kPa for 30 min when the chitosan microcarriers 0.1 g were soaked in 10 ml phosphate buffer solution (PBS, 0.05 M NaH2 PO4 –Na2 HPO4 , pH 7.2). The size was measured with Laser Diffraction Particle Size Analyzer SALD-3101 (Shimadzu, Japan). The water uptake of chitosan microcarriers was determined by measuring the weight of beads before and after soaking 0.10 g microcarriers in 25 ml PBS buffer for 24 h at 20 ◦ C and expressed in % of the initial bead weight. To obtain the weight after soaking,
Chitosan microcarriers (0.1 g) were soaked in 10 ml distilled water for 5 h to equilibrate moisture uptake, filtered and dried on filter paper. The resulting chitosan microcarriers were soaked in 2 ml BSA solution (2.5%, v/v) at 4 ◦ C for 1, 3, 6, 15 and 24 h and filtered through glass wool, respectively. The protein adsorptive capacity on the microcarriers (Pa%) was calculated using the following equation [18]: Pa% = (C1 V1 − C2 V2 )W −1 × 100%
(1)
where C1 and C2 are the protein concentration of the solution before and after adsorption, respectively; V1 and V2 are the volume of the solution before and after adsorption, respectively; and W is the weight of the chitosan microcarriers. The protein was determined by the Lowry method [19]. 2.5. Cell culture The specimens of mouse and human skins, obtained from Prof. Zhang at the Qingdao Hospital (Qingdao, China), were rinsed with D-Hanks solution supplemented with penicillin (1000 U/ml solution) and gentamicin (350 U/ml solution). Each treated skin was minced and explanted into tissue culture flasks (25 ml), incubated at 37 ◦ C, 5% CO2 , and 100% relative humidity [20]. The culture medium was RPMI-1640 supplemented with BSA (10%, v/v), penicillin (200 U/ml culture media) and streptomycin (200 U/ml culture media). After 15–20 days, the fibroblasts were subcultured with 0.01% EDTA-0.125% trypsin for the secondary culture. After being subcultured 2–3 times, epithelial cells were disappeared and only fibroblasts were present. The added amount of chitosan microcarriers in culture solution was 1 mg/ml. 2.6. Cytotoxicity test The general cytotoxicity test followed the method of Chen et al. [21]. Cells were inoculated into 96 well plates in 100 l RPMI-1640 medium with 10% BSA, each well containing 1 × 104 cells. Chitosan microcarriers were dried, ground into powder, and suspended in the medium at 3 mg/ml. The negative control was surgery seam (30 mg/10 ml medium); positive control was phenol (500 g/l) [21]. The suspension sample (10 l) was added to a well (five replicates per sample) and incubated for 2, 4 and 7 days. At the end of each exposure period, MTT (20 l) was added to each well and the cultures were incubated at 37 ◦ C for additional 3 h. The cells were then washed gently with PBS of pH 7.5 to remove untransformed MTT and sample residues. DMSO (150 l) was subsequently added to each well to dissolve the MTT
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Fig. 1. The scanning electron micrographs of the smooth-surface chitosan microcarriers. (a) The whole microcarriers. (b) The surface of the bead.
formazan purple crystals. The dissolution of the purple crystals was hastened by agitation of the solution by suction in and out of a pipette. Absorbency of the solution was measured at 540 nm using a UV spectrophotometer. Percentage of control (%) absorbance(bead samples) = 100 × absorbance(control) An increase in cell viability was verified for its statistical significance using an unpaired t-test (SigmaStatTM 2.0, Sachs, 1993) at p < 0.05 [22].
3. Results and discussion 3.1. General characteristics of chitosan microcarriers The fresh chitosan microcarriers prepared by the emulsion method showed smooth surface as revealed by SEM (Fig. 1). But in the process of drying, dewater directly in air often result in structural collapse and rupture of the microcarriers, and spherical microcarriers were obtained when ethanol was used as the syneresis agent. Without use of ethanol, the rate of coagulation was too slow to permit the maintenance of the spherical shape with concurrent bead consolidation. Therefore, ethanol was found to be a suitable coagulant and used for the preparation of smooth-surface chitosan beads. The chitosan microcarriers were semi-transparent (visual observation) and the diameter was in the range of 100–150 m (n = 5) (Fig. 2a). This range of size is suitable for microcarriers to be as cell carrier in the cell culture. Fig. 3 shows the FTIR spectra of chitosan and chitosan microcarriers. There were shifts in the amide I and II bands at 1657 and 1565/cm, respectively, indicating significant deacetylation of the chitosan molecule. At the same wavelengths of the FTIR spectrum of chitosan microcarriers, the shift I was weak, and it indicating the occurrence of the cross-linking reaction on the amino groups of chitosan molecules and a band was appeared at 1600/cm. Chitosan microcarriers remained structurally stable in PBS with the treatment at pH 7.2, temperature 120 ◦ C
Fig. 2. The size distributions of chitosan microcarriers. (a) Before soaking in buffer. (b) After soaking in buffer.
and pressure 150 kPa. After treatment in the PBS for 24 h, the chitosan microcarriers swelled to 150–200 m (n = 5) in size (Fig. 2b), the water uptake was 5.1 g PBS/1 g bead, and the weight increase of 48% ±0.5 (n = 5).
Fig. 3. The FTIR spectrums of chitosan and the chitosan microcarriers. (a) Chitosan. (b) Chitosan microcarriers.
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Fig. 4. The affinity of the chitosan microcarriers to bovine serum proteins in buffer of pH 7.2. Fig. 5. The adhering efficiency of fibroblast on smooth-surface chitosan microshperes.
3.2. Molecular affinity Chitosan microcarriers showed strong adsorption to bovine serum albumin (BSA) at pH 7.2 (2.5 %, v/v). As shown in Fig. 4, a rapid adsorption took place in the first 1 h and thereafter a steady rate was noted. The microcarriers adsorptive capacity reached 6.8 mg protein/g chitosan microcarriers at 24 h. Such protein binding suggests that smooth-surface chitosan microcarriers have bioaffinity, and the similar results were also reported in our previous study of the chitosan membrane [18]. This is believed to be an important property for
the material to have good biocompatibility being as a wounddressing biomaterial. 3.3. Cytotoxicity test on human and mouse fibroblast The chitosan microcarriers were ground into powder and suspended in culture medium to evaluate the cytotoxicity of the bead material. Table 1 shows the cytotoxicity response of human and mouse fibroblast cells to the chitosan microcarriers. In the MTT method, the concentration of formazan generation is directly proportional to the cytoviability. This
Fig. 6. The micrographs of chitosan microcarriers in the different growth progression of the human skin fibroblast on the bead surface. (a) The optical micrograph of the fibroblast on 24 h incubation. (b) The optical microscope observation of the fibroblast on 120 h incubation. (c) The SEM of the fibroblast on 24 h incubation. (d) The SEM of the fibroblast on 120 h incubation.
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Table 1 Relative growth rate (RGR) of cells Time
2 days 4 days 7 days a b c * **
RGR (%) Control
Negative controla
Positive controla
HMSFb
MSFc
100 100 100
108.21 127.62 128.66
20.00* 22.36** 14.21**
113.14** 131.04** 136.11**
115.88** 142.88** 137.24**
From mouse skin fibroblast. Human skin fibroblast. Mouse skin fibroblast. 0.01 < p < 0.05. p < 0.01.
is because the reduction of MTT into formazan reflects the action of mitochondrial enzymes in metabolically active cells. The concentration of formazan was measured spectrophotometrically [21]. The chitosan microcarriers were found to have no cytotoxicity to both human and mouse fibroblasts. The absorbency of the test groups remained high, approximately 113.14–142.89% of control groups, in the test period of 4 days. The results show that chitosan microcarriers are biocompatible in vitro, and should not impede cell growth, if it was used to be as a dressing material. 3.4. Cell growth on the chitosan microcarriers The chitosan microcarriers (100–200 m) could be suspended in the cell culture solution with a slight stirring (15 rpm). After cell inoculation and upon incubating 10 h, 76% cells adhered on the surface of chitosan microcarriers (Fig. 5). At incubation 24 h, the cells began to grow (Fig. 6a and c), and at 72 or 120 h, the cell grew in full bead and completely covered the surface of the microcarriers. The density of cell with the culture of chitosan microcarriers at 72 h was 8.0 × 105 cell/ml (n = 5). The high cell density on the surface of chitosan microcarriers at 120 h were show in Fig. 6b and d. These results support that the chitosan microcarriers had good affinity and has no cytotoxicity to the human skin fibroblast.
4. Conclusion The smooth-surface chitosan microcarriers in the size of 100–200 m can be prepared by the combination of emulsification and ethanol coagulation. The microcarriers were stable in PBS with the treatment at pH 7.2, temperature 120 ◦ C and pressure 150 kPa. The chitosan microcarriers had no cytotoxicity to both human and mouse fibroblasts. Chitosan microcarriers showed good binding to BSA at pH 7.2. Human skin fibroblast can well adhere on the surface of the microcarriers and grew to a high cell density. This shows that the smooth-surface chitosan microcarriers had good affinity and had no cytotoxicity to the human skin fibroblast. The results support that the smooth-surface chitosan microcarriers have good potential as to be a new wound-dressing biomaterial.
Acknowledgements We acknowledge the National Natural Science Foundation of China (30370344) and the Ministry of Education of China (2003-406) for financial support.
References [1] Y.N.L.B. Muhammad, L.Y. Lim, E. Khor, Preparation and characterization of chitin beads as a wound dressing precursor, J. Biomed. Mater. Res. 54 (2001) 59–68. [2] N.V. Majeti, Ravi Kumar, A review of chitin and chitosan applications, React. Funct. Polym. 46 (2000) 1–27. [3] M. Yoshikawa, T. Otsuki, T. Midorikawa, T. Terashi, Process for producing articles of regenerated chitin-chitosan containing material and the resulting articles. European Patent No. 794223, 1997. [4] T. Nakade, H. Yokota, H. Taniyama, Y. Hori, N. Agata, T. Ikeda, H. Furusaki, Y. Yamada, Y. Uchida, Matrix metalloproteinase (MMP) 9 induced in skin and subcutaneous tissue by implanted chitin in rats, Carbohydr. Polym. 41 (2000) 327–329. [5] Y. Ohshima, K. Nishino, Y. Yonekura, S. Kishimoto, S. Wakabayashi, Clinical application of chitin non-woven fabric as wound dressing, Eur. J. Surg. 10 (1987) 66–69. [6] H. Yokota, Chitin microparticles and their manufacture, Chem. Abstr. Jpn. Kokai Tokkyo Koho. Japanese Patent No. 63179901, 1987. [7] Y. Kawamura, M. Kinoshita, Y. Ito, E. Hirose, M. Itoyama, K. Ueda, Manufacture of chitin microcarriers, Chem. Abstr. Jpn. Kokai Tokkyo Koho. Japanese Patent No. 62100534, 1985. [8] H.E. Humphries, M. Christodoulides, J.E. Heckels, Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag, Protein Express. Purif. 26 (2002) 243–248. [9] B. Kerstin, L. peter, D. beate, P. Andreas, Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: Generic, parallel, and scalable protein purification, Protein Express. Purif. 24 (2002) 313–322. [10] J.E. Rebers, J.H. Willis, A conserved domain in arthropod cuticular proteins binds chitin, Insect Biochem. Mol. Biol. 31 (2001) 1083–1093. [11] X.F. Zeng, E. Ruckenstein, Macroporous chitin affinity membranes for wheat germ agglutinin purification from wheat germ, J. Membrane Sci. 156 (1999) 97–107. [12] M. Poirier, G. Charlet, Chitin fractionation and characterization in N,N-dimethylacetamide/lithium chloride solvent system, Carbohrdr. Polym. 50 (2002) 363–370. [13] L. Ilum, I. Jabbal-Gill, M. Hinchcliffe, A.N. Fisher, S.S. Davis, Chitosan as a novel nasal delivery system for vaccines, Adv. Drug Deliv. Rev. 51 (2001) 81–96.
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[14] P. He, S.S. Davis, L. Ilum, Chitosan microshperes prepared by spray drying, Int. J. Pharm. 187 (1999) 53–65. [15] G.A.F. Roberts, K.E. Taylor, Chitin chitosan: sources, chemistry, biochemistry, physical properties and applications, in: 4th International Conference, London, 1989, pp. 577–583. [16] X.G. Chen, W.S. Liu, G.H. Lang, C.S. Liu, R.S. Cong, Manufacture of chitin microshpheres, Chinese Patent No. ZL 96115714.3, 2002. [17] Y. Shigemasa, H. Matsuura, H. Sashiwa, H. Saimoto, Evaluation of different absorbance ratios from infrared spectroscopy for analyzing the degree of deacetylation in chitin, Int. J. Biol. Macromol. 18 (1996) 237–242. [18] X.G. Chen, L. Zheng, Z. Wang, C.Y. Lee, H.J. Park, Molecular affinity and permeability of different molecular weight chitosan membranes, J. Agric. Food Chem. 50 (2002) 5915–5918.
[19] Y. Sanz, S. Fadda, G. Vignolo, M.C. Aristoy, G. Oliver, F. Toldra, Hydrolytic action of Lactobacillus casei CRL 705 on pork muscle sarcoplasmic and myofibrillar proteins, J. Agric. Food Chem. 47 (1999) 3441–3448. [20] X.G. Chen, Z. Wang, W.S. Liu, H.J. Park, The effect of carboxymethyl-chitosan on proliferation and collagen secretion of normal and keloid skin fibroblasts, Biomaterials 23 (2002) 4609–4614. [21] X.G. Chen, Z. Wang, W.S. Liu, C.G. Liu, G.H. Lang, C.S. Liu, Cytotoxicity of 6-O-carboxymethyl-chitosan, J. Ocean Univ. Qingdao 30 (2000) 221–224. [22] L. Sachs, Statistische Methoden—Planung und Auswertung, Springer-Verlag, Berlin, Heidelber, New York, 1993, pp. 312.
Preparation and biocompatibility of chitosan microcarriers as biomaterial Xi-Guang Chen a,∗ , Cheng-Sheng Liu a , Chen-Guang Liu a , Xiang-Hong Meng a , Chong M. Lee b , Hyun-Jin Park c b
a The College of Marine Life Science, Ocean University of China, 5# Yushan Road Qingdao, Qingdao 266003, PR China The FSN Research Center, Department of Nutrition and Food Sciences, University of Rhode Island, Kingston RI 02881, USA c The Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea
Received 20 October 2004; received in revised form 4 July 2005; accepted 3 August 2005
Abstract Chitosan microcarriers (100–200 m) were prepared by the methods of emulsification and ethanol coagulant. It has smooth surface and was stable in phosphate buffer solution (PBS) of at pH 7.2 in the treatment of temperature 120 ◦ C and pressure 150 kPa. The chitosan microcarriers showed molecular affinity to the bovine serum proteins at pH 7.2. The adsorptive capacity of the microcarriers to the serum albumin was 6.8 mg protein/g chitosan bead. The chitosan microcarriers were found to have good biocompatibility and no cytotoxicity to both human and mouse fibroblasts in tissue cell culture. The fibroblasts well adhered on the smooth surface of the chitosan microcarriers and grew in high cell density. The results suggest a good potential of the chitosan microcarriers as a wound-healing biomaterial. © 2005 Elsevier B.V. All rights reserved. Keywords: Affinity; Microcarrier; Tissue cell culture; Adsorption
1. Introduction Chitosan (-1,4-linked-2-amino-2-deoxy-d-glucopyranose), made from deacetylated chitin (-1,4-linked-2acetamido-2-deoxy-d-glucose) is a polysaccharide of considerable promise in the field of biomedical research. Their biodegradability, biocompatibility, and nontoxicity allow widespread applications in wound healing [1]. The ability of chitosan and chitin to form gels, films, fibers, and sponges demonstrate their inherent versatility [2–4]. Chitin beads are potentially useful as a wound dressing material [1]. Chitin-based wound dressing materials promote and accelerate wound healing. Chitin may possess a tissue cell growth function and may action as a favorable scaffold for cell attachment and proliferation. This promotes rapid dermal regeneration allow accelerated wound healing [5]. The formation of chitin beads has been extensively investigated. Methods to prepare chitin beads include emulsifi∗
Corresponding author. Tel.: +86 532 82032586; fax: +86 532 82032586. E-mail address: [email protected] (X.-G. Chen).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.08.021
cation, suspension and homogenization, freeze-drying and hammer milling [6–11]. The water insolubility of chitin is a limitation for the preparation process of the beads, some special solvents or compounds, such as hexafluoroisopropanol, hexafluoroacetone, chloroalcohols, need to be added into the solvent in order to make chitin solutions [2,12]. Chitosan is soluble in the diluted acetic acid solution, and can be made into microcarriers. Chitosan microcarriers were found to have more utility in drug carrier and delivery systems [13]. The preparation of chitosan microcarriers involves the crushing and precipitation of chitosan solution droplets in an anionic coagulant such as alginate, tripolyphosphate [14]. The emulsion process had been employed for the preparation of chitosan beads with smooth surface and uniform size distribution [15]. The chitosan microcarriers with smoothsurface are thought to be good condition for cells adherence and growth on it. The characteristics and biocompatibility of the chitosan microcarriers are necessary to be described. In this paper, a smooth-surface chitosan microcarriers were prepared by emulsion and cross-linking method. The characteristics, bio-affinity to bovine serum albumin, and
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cytotoxicity of the chitosan microcarriers were investigated. The cell growth status on the chitosan microcarriers in vitro was described.
the chitosan microcarriers were filtered and blotted dry on filter paper (Xinhua 1# Filter paper). 2.4. Affinity experiment
2. Materials and methods 2.1. Materials Chitosan (MW 480,000 Da, DD 90%) was made from crab-shell and obtained from Biochemical Medicine Plant of Qingdao (Qingdao, China). Bovine serum albumin (BSA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (Sigma Co., St. Louis, MO, USA). 2.2. Chitosan bead preparation The preparation of chitosan microcarriers were followed a patented procedure [16]. Chitosan powder was dissolved in 1% acetic acid to give a 2.5% chitosan solution. The solution was filtered through glass wool. Filtered chitosan solution (50 ml), tween-80 (2.5 ml) and toluene (150 ml) were added into a 500 ml flask. The mixture solution were stirred 30 min with an electromagnetic bar to form an emulsion. Formalin (5 ml) and glutaraldehyde (1 ml) were added dropwise into the emulsion and were keeping in stir for 1 h. The mixture was filtered through 200-mesh nylon screen to separate the microcarriers, and the microcarriers were suspended in1000 ml distilled water (containing 0.05 g NaH4 B in water) for 12 h. The microcarriers were filtered and rinsed in distilled water, then successively dewatered in 30, 50, 80, 95 and 100% ethanol and ether, respectively. 2.3. Characterization of chitosan microcarriers Scanning electron micrographs (SEM) were obtained with a Stereoscan 250 Mk3 (Cambridge, UK). Chitosan microcarriers were gold-coated in a JEOL JFC-1100 ion-sputter. The IR spectrum of chitosan and chitosan microcarriers were recorded on an FT/IR-430 Fourier Transform Infrared Spectrometer (Jasco Co., Tokyo, Japan) at room temperature based on the method of Shigemasa [17]. The samples were 2 mg in the 100 mg KBr to be made pellet [17]. Thermal stability of chitosan microcarriers was assessed by checking the numbers of the intact microcarrier beads after heating at 120 ◦ C under pressure of 150 kPa for 30 min when the chitosan microcarriers 0.1 g were soaked in 10 ml phosphate buffer solution (PBS, 0.05 M NaH2 PO4 –Na2 HPO4 , pH 7.2). The size was measured with Laser Diffraction Particle Size Analyzer SALD-3101 (Shimadzu, Japan). The water uptake of chitosan microcarriers was determined by measuring the weight of beads before and after soaking 0.10 g microcarriers in 25 ml PBS buffer for 24 h at 20 ◦ C and expressed in % of the initial bead weight. To obtain the weight after soaking,
Chitosan microcarriers (0.1 g) were soaked in 10 ml distilled water for 5 h to equilibrate moisture uptake, filtered and dried on filter paper. The resulting chitosan microcarriers were soaked in 2 ml BSA solution (2.5%, v/v) at 4 ◦ C for 1, 3, 6, 15 and 24 h and filtered through glass wool, respectively. The protein adsorptive capacity on the microcarriers (Pa%) was calculated using the following equation [18]: Pa% = (C1 V1 − C2 V2 )W −1 × 100%
(1)
where C1 and C2 are the protein concentration of the solution before and after adsorption, respectively; V1 and V2 are the volume of the solution before and after adsorption, respectively; and W is the weight of the chitosan microcarriers. The protein was determined by the Lowry method [19]. 2.5. Cell culture The specimens of mouse and human skins, obtained from Prof. Zhang at the Qingdao Hospital (Qingdao, China), were rinsed with D-Hanks solution supplemented with penicillin (1000 U/ml solution) and gentamicin (350 U/ml solution). Each treated skin was minced and explanted into tissue culture flasks (25 ml), incubated at 37 ◦ C, 5% CO2 , and 100% relative humidity [20]. The culture medium was RPMI-1640 supplemented with BSA (10%, v/v), penicillin (200 U/ml culture media) and streptomycin (200 U/ml culture media). After 15–20 days, the fibroblasts were subcultured with 0.01% EDTA-0.125% trypsin for the secondary culture. After being subcultured 2–3 times, epithelial cells were disappeared and only fibroblasts were present. The added amount of chitosan microcarriers in culture solution was 1 mg/ml. 2.6. Cytotoxicity test The general cytotoxicity test followed the method of Chen et al. [21]. Cells were inoculated into 96 well plates in 100 l RPMI-1640 medium with 10% BSA, each well containing 1 × 104 cells. Chitosan microcarriers were dried, ground into powder, and suspended in the medium at 3 mg/ml. The negative control was surgery seam (30 mg/10 ml medium); positive control was phenol (500 g/l) [21]. The suspension sample (10 l) was added to a well (five replicates per sample) and incubated for 2, 4 and 7 days. At the end of each exposure period, MTT (20 l) was added to each well and the cultures were incubated at 37 ◦ C for additional 3 h. The cells were then washed gently with PBS of pH 7.5 to remove untransformed MTT and sample residues. DMSO (150 l) was subsequently added to each well to dissolve the MTT
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Fig. 1. The scanning electron micrographs of the smooth-surface chitosan microcarriers. (a) The whole microcarriers. (b) The surface of the bead.
formazan purple crystals. The dissolution of the purple crystals was hastened by agitation of the solution by suction in and out of a pipette. Absorbency of the solution was measured at 540 nm using a UV spectrophotometer. Percentage of control (%) absorbance(bead samples) = 100 × absorbance(control) An increase in cell viability was verified for its statistical significance using an unpaired t-test (SigmaStatTM 2.0, Sachs, 1993) at p < 0.05 [22].
3. Results and discussion 3.1. General characteristics of chitosan microcarriers The fresh chitosan microcarriers prepared by the emulsion method showed smooth surface as revealed by SEM (Fig. 1). But in the process of drying, dewater directly in air often result in structural collapse and rupture of the microcarriers, and spherical microcarriers were obtained when ethanol was used as the syneresis agent. Without use of ethanol, the rate of coagulation was too slow to permit the maintenance of the spherical shape with concurrent bead consolidation. Therefore, ethanol was found to be a suitable coagulant and used for the preparation of smooth-surface chitosan beads. The chitosan microcarriers were semi-transparent (visual observation) and the diameter was in the range of 100–150 m (n = 5) (Fig. 2a). This range of size is suitable for microcarriers to be as cell carrier in the cell culture. Fig. 3 shows the FTIR spectra of chitosan and chitosan microcarriers. There were shifts in the amide I and II bands at 1657 and 1565/cm, respectively, indicating significant deacetylation of the chitosan molecule. At the same wavelengths of the FTIR spectrum of chitosan microcarriers, the shift I was weak, and it indicating the occurrence of the cross-linking reaction on the amino groups of chitosan molecules and a band was appeared at 1600/cm. Chitosan microcarriers remained structurally stable in PBS with the treatment at pH 7.2, temperature 120 ◦ C
Fig. 2. The size distributions of chitosan microcarriers. (a) Before soaking in buffer. (b) After soaking in buffer.
and pressure 150 kPa. After treatment in the PBS for 24 h, the chitosan microcarriers swelled to 150–200 m (n = 5) in size (Fig. 2b), the water uptake was 5.1 g PBS/1 g bead, and the weight increase of 48% ±0.5 (n = 5).
Fig. 3. The FTIR spectrums of chitosan and the chitosan microcarriers. (a) Chitosan. (b) Chitosan microcarriers.
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Fig. 4. The affinity of the chitosan microcarriers to bovine serum proteins in buffer of pH 7.2. Fig. 5. The adhering efficiency of fibroblast on smooth-surface chitosan microshperes.
3.2. Molecular affinity Chitosan microcarriers showed strong adsorption to bovine serum albumin (BSA) at pH 7.2 (2.5 %, v/v). As shown in Fig. 4, a rapid adsorption took place in the first 1 h and thereafter a steady rate was noted. The microcarriers adsorptive capacity reached 6.8 mg protein/g chitosan microcarriers at 24 h. Such protein binding suggests that smooth-surface chitosan microcarriers have bioaffinity, and the similar results were also reported in our previous study of the chitosan membrane [18]. This is believed to be an important property for
the material to have good biocompatibility being as a wounddressing biomaterial. 3.3. Cytotoxicity test on human and mouse fibroblast The chitosan microcarriers were ground into powder and suspended in culture medium to evaluate the cytotoxicity of the bead material. Table 1 shows the cytotoxicity response of human and mouse fibroblast cells to the chitosan microcarriers. In the MTT method, the concentration of formazan generation is directly proportional to the cytoviability. This
Fig. 6. The micrographs of chitosan microcarriers in the different growth progression of the human skin fibroblast on the bead surface. (a) The optical micrograph of the fibroblast on 24 h incubation. (b) The optical microscope observation of the fibroblast on 120 h incubation. (c) The SEM of the fibroblast on 24 h incubation. (d) The SEM of the fibroblast on 120 h incubation.
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Table 1 Relative growth rate (RGR) of cells Time
2 days 4 days 7 days a b c * **
RGR (%) Control
Negative controla
Positive controla
HMSFb
MSFc
100 100 100
108.21 127.62 128.66
20.00* 22.36** 14.21**
113.14** 131.04** 136.11**
115.88** 142.88** 137.24**
From mouse skin fibroblast. Human skin fibroblast. Mouse skin fibroblast. 0.01 < p < 0.05. p < 0.01.
is because the reduction of MTT into formazan reflects the action of mitochondrial enzymes in metabolically active cells. The concentration of formazan was measured spectrophotometrically [21]. The chitosan microcarriers were found to have no cytotoxicity to both human and mouse fibroblasts. The absorbency of the test groups remained high, approximately 113.14–142.89% of control groups, in the test period of 4 days. The results show that chitosan microcarriers are biocompatible in vitro, and should not impede cell growth, if it was used to be as a dressing material. 3.4. Cell growth on the chitosan microcarriers The chitosan microcarriers (100–200 m) could be suspended in the cell culture solution with a slight stirring (15 rpm). After cell inoculation and upon incubating 10 h, 76% cells adhered on the surface of chitosan microcarriers (Fig. 5). At incubation 24 h, the cells began to grow (Fig. 6a and c), and at 72 or 120 h, the cell grew in full bead and completely covered the surface of the microcarriers. The density of cell with the culture of chitosan microcarriers at 72 h was 8.0 × 105 cell/ml (n = 5). The high cell density on the surface of chitosan microcarriers at 120 h were show in Fig. 6b and d. These results support that the chitosan microcarriers had good affinity and has no cytotoxicity to the human skin fibroblast.
4. Conclusion The smooth-surface chitosan microcarriers in the size of 100–200 m can be prepared by the combination of emulsification and ethanol coagulation. The microcarriers were stable in PBS with the treatment at pH 7.2, temperature 120 ◦ C and pressure 150 kPa. The chitosan microcarriers had no cytotoxicity to both human and mouse fibroblasts. Chitosan microcarriers showed good binding to BSA at pH 7.2. Human skin fibroblast can well adhere on the surface of the microcarriers and grew to a high cell density. This shows that the smooth-surface chitosan microcarriers had good affinity and had no cytotoxicity to the human skin fibroblast. The results support that the smooth-surface chitosan microcarriers have good potential as to be a new wound-dressing biomaterial.
Acknowledgements We acknowledge the National Natural Science Foundation of China (30370344) and the Ministry of Education of China (2003-406) for financial support.
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