Trends Biomater. Artif. Organs, 22(2), pp 111-115 Thin (2008) Chitin Vol Nanofibre Reinforced Chitosan Films for Wound Healing Application
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Chitin Nanofibre Reinforced Thin Chitosan Films for Wound Healing Application Shelma R., Willi Paul and Sharma C.P.* Division of Biosurface Technology, Biomedical Technology Wing Sree Chitra Tirunal Institute for Medical Sciences & Technology Thiruvananthapuram 695 012 *Corresponding author e-mail:
[email protected] Received 26 June 2008; published online 1 August 2008 Both chitin and chitosan possess many properties that are advantageous for wound healing like biocompatibility, biodegradability, hemostatic activity, healing acceleration, non-toxicity, adsorption properties and anti infection properties. However, pure chitosan films have poor tensile strength and elasticity. Hence development of high strength composites that are biocompatible and that can help in wound healing may be necessary for wound dressing applications. An attempt has been made to develop a composite film from chitosan by incorporating chitin nanofibres to improve its tensile strength and elasticity. Nanocomposite films were prepared from chitosan by solution casting after incorporating chitin nanofibres as nanofillers. Present study suggests that the tensile strength of the chitosan films can be increased up to a significant level by incorporating chitin nanofibres without appreciable change in water vapor permeability. © Society for Biomaterials and Artificial Organs (India), 20080626-22.
Introduction Animals and plants synthesize biocomposites with high strength consisting of fibrous biopolymers. Classic example is cellulose which consists of whisker like micro-fibrils that are synthesized and deposited in a definite manner which imposes high strength. Living tissues are composites themselves with a number of levels of hierarchy. Reinforcement of polymer with nanosized particles or fibers is a promising technique that is capable of yielding materials with enhanced performance but without involvement of expensive synthesis procedures. Chitin, an extracted component of crustacean exoskeleton, is the most abundant polysaccharide in nature in addition to starch and cellulose. Whiskers or nano-fibres have been prepared from chitin by many investigators
(1). The derivatives of chitin have many properties that make them attractive for a wide range of applications from biomedicine, agricultural and cosmetics. Antibacterial, antifungal and antiviral properties make them particularly useful for biomedical applications such as wound dressings, surgical sutures, as aids in cataract surgery, periodontal disease treatment, etc. Research has shown that chitin and chitosan are nontoxic and non-allergic so that the body will not reject these compounds. Both chitin and chitosan possess many properties that are advantageous for wound healing like biocompatibility, biodegradability (2), hemostatic activity (3), healing acceleration, non-toxicity, adsorption properties and anti infection properties (4-6). An effective wound dressing not only protects the wound from its surroundings but also promotes the wound healing by providing an optimum microenvironment for healing, removing any
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excess wound exudates and allowing continuous tissue reconstruction. Mechanical property is one of the critical and important characteristic of a wound dressing. Chitosan has been studied as an excellent wound dressing film. However, pure chitosan films have poor tensile strength and elasticity. Hence development of high strength composites that are biocompatible and that can help in wound healing may be necessary for wound dressing applications. An attempt has been made to develop a composite film from chitosan by incorporating chitin nanobibres to improve its tensile strength and elasticity. Nanocomposite films were prepared from chitosan by solution casting after incorporating chitin nanofibres as nanofillers. Its mechanical strength, swelling characteristics and water vapour transmission rates were studied. Materials and Methods Chitosan (degree of deacetylation of 87%, molecular weight of 270kD) was obtained from Central Institute of Fisheries Technology, Cochin, India. Chitin purified powder was obtained from SD Fine Chem India Ltd., Mumbai India. All other chemicals are of analytical reagent grade. Preparation of chitin whisker Chitin nanofibres were prepared as per the reported procedure (7). Briefly, purified chitin powder was hydrolysed by adding 3N HCl under stirring for 2h at 104ºC. HCl to chitin ratio of was maintained at 30ml/g. After hydrolysis, suspensions were diluted with distilled water followed by centrifugation at 9500 rpm for 10min and this process was repeated thrice. The suspension was then transferred to a dialysis bag and dialyzed against distilled water overnight. The pH of the suspension was adjusted to 2.5 by adding HCl. This was subjected to ultrasonication for 20min at 42W. This suspension displayed a colloidal behavior whose stability was attributed to the presence of NH3+at the surface of crystallites resulting from the protonation of amino group. Preparation of chitin reinforced chitosan film Chitin nanofibres in varying amounts, i.e. in the
range of 0-37.5%, was added to different batches of chitosan solution and stirred for 2h. This was then cast on a glass plate and dried in an oven at 37ºC. The films were neutralized by the addition of NaOH and washed thoroughly with distilled water. Washed films were then dried at the room temperature. The composite films are designated as CH-W1, CH-W2, CHW3, CH-W4, CH-W5 and CH-W6 with chitin nanofibre content of 2.7%, 10%, 14%, 17% 31.5% and 37.5% respectively. Characterization of chitosan nanocomposite films Chitin nanofibres were subjected to dynamic light scattering studies to evaluate its particle size utilizing a zetasizer (Nano ZS, Malvern Instruments UK). Chitosan nanocomposite films were characterized by FTIR spectroscopy using Nicolet Impact 410 FTIR spectrometer. Degree of swelling of these films was evaluated after allowing it to swell in distilled water for 24 hours. Tensile properties of the films were evaluated using Universal Testing Machine with the load range 0.1kN, cross head speed of 10mm/min and a gauge length of 50mm at 23ºC. Films for the tests were cut with a dimension of 5mm x 90mm with the thickness about 28μm. Water vapor permeability of chitin reinforced composite films was evaluated as per ASTM standard E96. Films were sealed tightly to cup containing water. Initial weights were taken and the cups were kept in the oven at 37ºC. Sealed cups were weighed periodically after each 2h. Readings were noted for three days. Results and Discussion Chitin nanofibres prepared by a standard procedure was evaluated for its particle size. The particle size distribution is depicted in figure 1. Particle size distribution exhibited a bimodal curve with size varying from 15nm to 400nm with majority between 20nm and 300nm. As reported earlier the chitin whiskers had an average length of 300nm and diameter of 20nm observed through transmission electron microscopy (8). From the particle size distribution by dynamic light scattering it seems
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that it is almost similar to the reported values. Chitin forms microfibrilar arrays in living organisms (9) which are aligned in larger dimension fibers with high degree of orientation that provides strength to the system. Therefore these chitin nanofibres can be used as fillers in polymers to prepare high strength materials.
Fig. 1: Particle size distribution of chitin nanofibres by dynamic light scattering
FTIR spectra of all samples included bands which can be observed at approximately 3435, 3370, 3295, 2929, 2884, 1651, 1548, 1418, 1379, 1321, 1260, 1153, 1072, 1027, 896 and 664 cm-1 that are consistent with the structure of chitosan (10). In addition to that, in the spectra of chitin reinforced films characteristic bands corresponding chitin were found at 972 cm-1 (CH3 vibration), and amide I band at 1623 cm-1 which increases with the chitin whisker content. The swelling percentage, mechanical data and water vapor transmission rate of nanocomposite films are given in table 1. As the nanofibre content increased the water swelling property of composite films decreased. The tensile strength of the composite film increased till a whisker content of 17%. Further increase in whisker content drastically decreased its tensile strength. However, there was no significant change in water vapor transmission rate of composite films. Faster wound healing was observed when a variety of chitosan-based skin graft material was tested in Guinea pigs and rabbits in our laboratory (11). A dressing with an optimal combination of chitosan, alginate and poly ethylene glycol containing a synergistic
Fig. 2: FT-IR spectra of chitosan and chitin nanofibre reinforced composite films. Arrows indicate peaks at 1623 cm -1 and 972 cm -1 characteristic of chitin
combination of an antibiotic and an analgesic was studied on human subjects with chronic non-healing ulcers. It was observed that this material made the ulcer cleaner and had beneficial effect in the control of infection (12). Application of this dressing was extremely
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Table 1: Tensile strength, Swelling percentage and Water vapor transmission rate (WVTR) of chitosan and chitin nanofibre reinforced chitosan composite films Sample Chitosan -CH CH-W1 CH-W2 CH-W3 CH-W4 CH-W5 CH-W6
Tensile Stress (MPa)
Tensile Strain (%)
Modulus (MPa)
Swelling (%)
WVTR 2 (g/m /hr)
45.36 ± 6.01 53.66 ± 6.93 52.65 ± 9.02 55.80 ± 4.99 59.50 ± 5.27 33.54 ± 3.04 25.14 ± 7.46
7.2 ± 1.3 7.37 ± 1.3 4.90 ± 1.05 4.41 ± 1.66 7.05 ± 1.81 2.89 ± 0.59 1.87 ± 0.69
1344 ± 316 2318 ± 619 2053 ± 327 1850 ± 388 1622 ± 377 1898 ± 228 2082 ± 319
99 45 62 56 47 25 14
57 ± 1 58 ± 2 57 ± 2 59 ± 2 58 ± 2 60 ± 2 65 ± 8
satisfactory particularly at donor site with painless healing. Conclusion Chitosan provides a non-protein matrix for 3D tissue growth and activates macrophages for tumoricidal activity. It stimulates cell proliferation and histoarchitectural tissue organization. Chitosan is a hemostat, which helps in natural blood clotting and blocks nerve endings reducing pain. Chitosan will gradually depolymerize to release N-acetyl-b-Dglucosamine, which initiates fibroblast proliferation and helps in ordered collagen deposition and stimulates increased level of natural hyaluronic acid synthesis at the wound site. It helps in faster wound healing and scar prevention. However, its mechanical strength should be increased further to facilitate practical applications as wound dressing films. Present study suggests that the tensile strength of the
chitosan films can be increased up to a significant level by incorporating chitin nanofibres without appreciable change in water vapor permeability. However, the percentage swelling of the composite chitosan films decreased with increase in chitin whisker content. Since the orientation of chitin nanofibres plays an important role in its mechanical strength, further studies are required to equate the orientation of chitin nanofibres and its effect on mechanical properties. Acknowledgment We are grateful to Prof. K. Mohandas, Director, and Dr. G.S. Bhuvaneshwar, Head BMT Wing of Sree Chitra Tirunal Institute for Medical Sciences & Technology for providing facilities for the completion of this work. This work has been partially funded by Department of Science & Technology, Govt. of India through the project FADDS #8013.
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19, 2003. 9. Extra Cellular Matrix of animal connective tissue in Molecular biology of the cell, Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Eds Garland Science, London, 1178-1195, 2007. 10. Acosta, C. Jiménez, V. Borau y A. Heras, Extraction and Characterization of Chitin from Crustaceans, Biomass and Bioenergy, 5, 145, 1993. 11. R.S. Jayasree, K. Rathinam, C.P. Sharma, Development of Artificial Skin (Template) and influence of different types of sterilization procedure on wound healing pattern in rabbits and guinea pigs, J. Biomater. Appl., 10, 144-153, 1995. 12. R. Nair, Report on the trial of chitosan film in chronic ulcers, 2000 (unpublished report).
