Abstract
In this work, chitosan-based films containing gelatin and chondroitin-4-sulfate (C4S) with and without ZnO particles were produced and tested in vitro to investigate their potential wound healing properties. Chitosans were produced from shrimp-head processing waste by alkaline deacetylation of chitin to obtain chitosans differing in molecular weight and degree of deacetylation (80 ± 0.5%). The film-forming solutions (chitosan, C4S and gelatin) and ZnO suspension showed no toxicity towards fibroblasts or keratinocytes. Chitosan was able to agglutinate red blood cells, and film-forming solutions induced no hemolysis. Film components were released into solution when incubated in PBS as demonstrated by protein and sugar determination. These data suggest that a stable, chitosan-based film with low toxicity and an ability to release components would be able to establish a biocompatible microenvironment for cell growth. Chitosan-based films significantly increased the percentage of wound healing (wound contraction from 65 to 86%) in skin with full-thickness excision when compared with control (51%), after 6 days. Moreover, histological analysis showed increased granulation tissue in chitosan and chitosan/gelatin/C4S/ZnO films. Chitosan-based biopolymer composites could be used for improved biomedical applications such as wound dressings, giving them enhanced properties.
Similar content being viewed by others
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.References
Cahú, T. B., Santos, S. D., Mendes, A., Córdula, C. R., Chavante, S. F., Carvalho Jr., L. B., Nader, H. B., & Bezerra, R. S. (2012). Recovery of protein, chitin, carotenoids and glycosaminoglycans from Pacific white shrimp (Litopenaeus vannamei) processing waste. Process Biochemistry, 47, 570–577.
Philibert, T., Lee, B. H., & Fabien, N. (2016). Current status and new perspectives on chitin and chitosan as functional biopolymers. Applied Biochemistry and Biotechnology, 1–24.
Weska, R. F., Moura, J. M., Batista, L. M., Rizzi, J., & Pinto, L. A. A. (2007). Optimization of deacetylation in the production of chitosan from shrimp wastes: use of response surface methodology. Journal of Food Engineering, 80, 749–753.
Croisier, F., & Jérôme, C. (2013). Chitosan-based biomaterials for tissue engineering. European Polymer Journal, 49, 780–792.
Šimkovic, I. (2008). What could be greener than composites made from polysaccharides? Carbohydr Polymer, 74, 759–762.
Jayakumar, R., Prabaharan, M., Kumar, P. S., Nair, S. V., & Tamura, H. (2011). Novel chitin and chitosan nanofibers in biomedical applications. Biotechnology Advances, 29, 322–337.
TVL, H.-B., Vidyavathi, M., Kavitha, K., Sastry, T. P., & Suresh-Kumar, R. V. (2010). Preparation and evaluation of chitosan gelatin composite films for wound healing activity. Biomaterials and Artificial Organs, 24, 123–130.
Ramasamy, P., & Shanmugam, A. (2015). Characterization and wound healing property of collagen–chitosan film from Sepia kobiensis (Hoyle, 1885). Int. J. Biol. Macromolec., 74, 93–102.
Lansdown, A. B., Mirastschijski, U., Stubbs, N., Scanlon, E., & Ågren, M. S. (2007). Zinc in wound healing: theoretical, experimental, and clinical aspects. Wound Repair and Regeneration, 15, 2–16.
Jhong, J. F., Venault, A., Liu, L., Zheng, J., Chen, S. H., Higuchi, A., Huang, J., & Chang, Y. (2014). Introducing mixed-charge copolymers as wound dressing biomaterials. ACS Applied Materials & Interfaces, 6, 9858–9870.
Campo, G. M., Avenoso, A., Campo, S., Ferlazzo, A. M., & Calatroni, A. (2006). Chondroitin sulphate: antioxidant properties and beneficial effects. Mini Reviews in Medicinal Chemistry, 6, 1311–1320.
Smith, M. M., & Melrose, J. (2015). Proteoglycans in normal and healing skin. Adv Wound Care, 1, 152–173.
Yamada, S., & Sugahara, K. (2008). Potential therapeutic application of chondroitin sulfate/dermatan sulfate. Current Drug Discovery Technologies, 5, 289–301.
Sezer, A.D. and Cevher, E. (2011) Biopolymers as wound healing materials: challenges and new strategies. In: Biomaterials applications for nanomedicine, (Pignatello R) InTech pp 384–414.
Roncal, T., Oviedo, A., Armentia, I. L., Fernandez, L., & Villaran, M. C. (2007). High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan. Carbohydrate Research, 342, 2750–2756.
Brugnerotto, J., Lizardi, J., Goycoolea, F. M., Argüelles-Monal, W., Desbrieres, J., & Rinaudo, M. (2001). An infrared investigation in relation with chitin and chitosan characterization. Polymer, 42, 3569–3580.
Patra, M. K., Manoth, M., Singh, V. K., Gowd, G. S., Choudhry, V. S., Vadera, S. R., & Kumar, N. (2009). Synthesis of stable dispersion of ZnO quantum dots in aqueous medium showing visible emission from bluish green to yellow. Journal of Luminescence, 129, 320–324.
Gaidamashvili, M., & van Staden, J. (2002). Interaction of lectin-like proteins of South African medicinal plants with Staphylococcus aureus and Bacillus subtilis. Journal of Ethnopharmacology, 80, 131–135.
RMPB, C., AFM, V., MLV, O., LCBB, C., MTS, C., & Carneiro-da-Cunha, M. G. (2011). A mew mistletoe Phthirusa pyrifolia leaf lectin with antimicrobial properties. Process Biochemistry, 45, 526–533.
Paula, A. M., DST, M., Araújo Jr., R. T., Souza Filho, A. G., & Alves, O. L. (2012). Suppression of the hemolytic effect of mesoporous silica nanoparticles after protein corona interaction: independence of the surface microchemical environment. Journal of the Brazilian Chemical Society, 23, 1807–1814.
Bradford, M. M. (1976). Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1958). Colorimetric method for determination of sugars and related substances. Anal Chem, 28, 360–356.
Dai, T., Tanaka, M., Huang, Y. Y., & Hamblin, M. R. (2011). Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Review of Anti-Infective Therapy, 9, 857–879.
Tanaka, A., Nagate, T., & Matsuda, H. (2005). Acceleration of wound healing by gelatin film dressings with epidermal growth factor. The Journal of Veterinary Medical Science, 67, 909–913.
Chong, E. J., Phan, T. T., Lim, I. J., Zhang, Y. Z., Bay, B. H., Ramakrishna, S., & Lim, C. T. (2007). Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution. Acta Biomaterialia, 3, 321–330.
Gilbert, M. E., Kirker, K. R., Gray, S. D., Ward, P. D., Szakacs, J. G., Prestwich, G. D., & Orlandi, R. R. (2014). Chondroitin sulfate hydrogel and wound healing in rabbit maxillary sinus mucosa. Laryngoscope, 114, 1406–1409.
Sezer, A. D., Hatipoglu, F., Cevher, E., Oğurtan, Z., Bas, A. L., & Akbuğa, J. (2007). Chitosan film containing fucoidan as a wound dressing for dermal burn healing: preparation and in vitro/in vivo evaluation. AAPS PharmSciTech, 8, 94–101.
Selvam, S., & Sundrarajan, M. (2012). Functionalization of cotton fabric with PVP/ZnO nanoparticles for improved reactive dye ability and antibacterial activity. Carbohyd. Polym., 87, 1419–1424.
Kim, M. S., Park, S. J., Gu, B. K., & Kim, C. H. (2015). Fabrication of chitosan nanofibers scaffolds with small amount gelatin for enhanced cell viability. In Applied Mechanics and Materials, 749, 220–224 Trans Tech Publications.
Hajji, S., Younes, I., Rinaudo, M., Jellouli, K., & Nasri, M. (2015). Characterization and in vitro evaluation of cytotoxicity, antimicrobial and antioxidant activities of chitosans extracted from three different marine sources. Applied Biochemistry and Biotechnology, 177, 18–35.
Roche, E. D., Renick, P. J., Tetens, S. P., & Carson, D. L. (2012). A model for evaluating topical antimicrobial efficacy against methicillin-resistant Staphylococcus aureus biofilms in superficial murine wounds. Antimicrobl Agents Chemother, 56, 4508–4510.
Ridolfi, D. M., Marcato, P. D., Machado, D., Silva, R. A., Justo, G. Z., & Durán, N. (2011). In vitro cytotoxicity assays of solid lipid nanoparticles in epithelial and dermal cells. Journal of Physics Conference Series, 304, 012032.
Subhasree, R. S., Selvakumar, D., & Kumar, N. S. (2012). Hydrothermal mediated synthesis of ZnO nanorods and their antibacterial properties. Lett. Appl. Nano. Bio. Sci., 1, 2–7.
Vasile, B. S., Oprea, O., Voicu, G., Ficai, A., Andronescu, E., Teodorescu, A., & Holban, A. (2014). Synthesis and characterization of a novel controlled release zinc oxide/gentamicin–chitosan composite with potential applications in wounds care. International Journal of Pharmaceutics, 463, 161–169.
Souza, A. P., Gerlach, R. F., & SRP, L. (2000). Inhibition of human gingival gelatinases (MMP-2 and MMP-9) by metal salts. Dental Materials, 16, 103–108.
Souza, A. P., Gerlach, R. F., & SRP, L. (2001). Inhibition of human gelatinases by metals released from dental amalgam. Biomaterials, 22, 2025–2030.
Sudesh Kumar, P. T., Lakshmanan, V. K., Anilkumar, T. V., Ramya, C., Reshmi, P., Unnikrishnan, A. G., Nair, S. V., & Jayakumar, R. (2012). Flexible and microporous chitosan hydrogel/nanoZnO composite bandages for wound dressing: in vitro and in vivo evaluation. ACS Applied Materials & Interfaces, 4, 2618–2629.
Wang, X., Du, Y., & Liu, H. (2004). Preparation, characterization and antimicrobial activity of chitosan–Zn complex. Carbohyd Polym., 56, 21–26.
Selvam, S., Rajiv-Gandhi, R., Suresh, J., Gowri, S., Ravikumar, S., & Sundrarajan, M. (2012). Antibacterial effect of novel synthesized sulfated cyclodextrin crosslinked cotton fabric and its improved antibacterial activities with ZnO, TiO2 and Ag nanoparticles coating. International Journal of Pharmaceutics, 434, 366–374.
Sudesh Kumar, P. T., Lakshmanan, V. K., Raj, M., Biswas, R., Hiroshi, T., Nair, S. V., & Jayakumar, R. (2013). Evaluation of wound healing potential of β-chitin hydrogel/nano zinc oxide composite bandage. Pharmaceutical Research, 30, 523–537.
Rao, S. B., & Sharma, C. P. (1997). Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. Journal of Biomedical Materials Research, 34, 21–28.
Paul, W., & Sharma, C. P. (2004). Chitosan and alginate wound dressings: a short review. Biomaterials and Artificial Organs, 18, 18–23.
Kong, M., Chen, X. G., Xing, K., & Park, H. J. (2010). Antimicrobial properties of chitosan and mode of action: a state of the art review. Inter. J. Food Microbiol., 44, 51–63.
Yang, T. C., Chou, C. C., & Li, C. F. (2005). Antibacterial activity of N-alkylated disaccharide chitosan derivatives. Inter. J. Food Microbiol., 97, 237–245.
Tompkins, G. G. and Burke, J. F. (1996). Alternative wound coverings. In: Total burn care (Herndon DN.) W.B. Saunders, PH, pp 164–172.
Pruitt, B. A., & Levine, N. S. (1984). Characteristics and uses of biological dressings and skin substitutes. Archives of Surgery, 119, 312–322.
Jones, I., Currie, L., & Martin, R. (2002). A guide to biological skin substitutes. Br. J. Plast. Sur., 55, 185–193.
Acknowledgments
The authors are thankful to the financing agencies CAPES and CNPq for funding this project; to “Financiadora de Estudos e Projetos”—FINEP, of the Brazilian government, responsible for funding “RECARCINA” (Project No. 01.13.0220.00); and to “Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco” FACEPE for granting the doctoral scholarship.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Supplementary Figure 1
(DOC 2102 kb)
Supplementary Figure 2
(DOC 356 kb)
Supplementary Figure 3
(DOC 106 kb)
Rights and permissions
About this article
Cite this article
Cahú, T.B., Silva, R.A., Silva, R.P.F. et al. Evaluation of Chitosan-Based Films Containing Gelatin, Chondroitin 4-Sulfate and ZnO for Wound Healing. Appl Biochem Biotechnol 183, 765–777 (2017). https://doi.org/10.1007/s12010-017-2462-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-017-2462-z