Differences between Solution and Membrane Forms of Chitosan on the In Vitro Activity of Fibroblasts

Abstract

Background: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. Aims: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. Study Design: In vitro study. Methods: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group); cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group) or chitosan solution at a concentration of 2.0% (Solution Group). Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1); and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2). Results: Scanning electron microscopic investigations of the experimental groups revealed cells with welldefined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10th day of culture for both methods. Bromodeoxyuridine (BrdU) and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5th and 10th days. For the second method, the membranous form on the 10th day and solution form on the 5th day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5th day of treatment with the membranous form, whereas cellular viability was highest in the solution form of method 2 on the 5th day. Conclusion: The membranous form of chitosan induced a significant proliferative effect and increased the ratio of cell-to-cell junctions of Swiss 3T3 mouse embryonic fibroblasts. Conveniently, the solution form also resulted in enhanced cell proliferation and viability compared to the control group. As the solution form is easy to prepare and apply to cells compared to the membrane form, the application of Chitosan directly to media appears to be a convenient alternative for tissue engineering approaches

Keywords

• Cell culture, cell viability, chitosa, collagen immunohistochemistry, scanning electron microscopy, Swiss 3T3

References

1. 1. Akbuga J. A Biopolymer: Chitosan. Int J Pharm 1995;1:3-18. 2. Koide S. Chitin-chitosan: properties, benefits and risks. Nutrition Research 1998;18:1091-1101. [CrossRef]
2. 3. Hwang SM, Chen CY. Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages. Biochem Biophys Res Commun 2000;271:229-33. [CrossRef]
3. 4. Yang TL. Chitin-based Materials in Tissue Engineering: Applications in Soft Tissue and Epithelial Organ. Int J Mol Sci 2011;12:1936-63. [CrossRef]
4. 5. Aral C, Akbuga J. Alternative approach to the preparation of chitosan beads. Int J Pharm 1998;168:9-15. [CrossRef]
5. 6. Wan Y, Wu H, Wen D. Porous-conductive chitosan scaffolds for tissue engineering, 1. Preparation and characterization. Macromol Biosci 2004;16:882-90. [CrossRef]
6. 7. Ueno H, Nakamura M, Murakami M. Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages. Biomaterials 2001;22:2125-30. [CrossRef]
7. 8. Pereira F, Pereira C, Lacerda MH. Contact dermatitis due to a cream containing chitin and a Carbitol. Contact Dermatitis 1998;38:290-1. [CrossRef]
8. 9. Stone CA, Wright H, Clarke T, Powell R, Devaraj VS. Healing skin graft donor sites dressed with chitosan. Brit J Plast Surg 2000;53:601-6. [CrossRef]

9. 10. Denuziere A, Ferrier D, Damour O, Domard A. Chitosan-chondroitin sulfate and chitosan-hyaluronate polyelectrolyte complexes: biological properties. Biomaterials 1998;19:1275-85. [CrossRef]
10. 11. Muzzarelli R, Baldassarre V, Conti F, Ferrara P, Biagini G, Gazzanelli G, et al. Biological activity of chitosan: ultrastructural study. Biomaterials 1988;9:247-52. [CrossRef]
11. 12. Tahara K, Sakai T, Yamamoto H, Takeuchi H, Hirashima N, Kawashima Y. Improvements in transfection efficiency with chitosan modified poly(DL-lactide-co-glycolide) nanospheres prepared by the emulsion solvent diffusion method, for gene delivery. Chem Pharm Bull 2011;59:298-301. [CrossRef]
12. 13. Diao ZY, Fu HL, Nie CL, Hao LJ, Yang DP, Chen WH. Controlled release of transforming growth factor-beta receptor kinase inhibitor from thermosensitive Chitosan-based hydrogel: application for prevention of capsular contracture. Chin Med J (Engl) 2011;124:284-90.
13. 14. Kong LJ, Ao Q, Xi J, Zhang L, Gong YD, Zhao NM, et al. Proliferation and differentiation of MC 3T3-E1 cells cultured on nanohydroxyapatite/chitosan composite scaffolds. Sheng Wu Gong Cheng Xue Bao 2007;23:262-7. [CrossRef]
14. 15. Aaronson SA, Todaro GJ. Development of 3T3-like lines from Balb/c mouse embryo cultures. Transformation susceptibility to SV40. J Cell Physiol 1968;72:141-8. [CrossRef]
15. 16. Chen RH, Tsaih ML. Effect of temperature on the intrinsic viscosity and conformation of chitosan in dilute HCI solution. Int J Biol Macromol 1998;23:135-41. [CrossRef]
16. 17. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;16:55-63. [CrossRef]
17. 18. Howling GI, Dettmar PW, Goddard PA, Hampson FC, Dornish M, Wood EJ. The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro. Biomaterials 2001;22:2959-66. [CrossRef]
18. 19. Senel S, McClure SJ. Potential applications of chitosan in veterinary medicine. Adv Drug Deliv Rev 2004;56:1467-80. [CrossRef]
19. 20. Kiyozumi T, Kanatani Y, Ishihara M, Saitoh D, Shimizu J, Yura H, et al.The effect of chitosan hydrogel containing DMEM/F12 medium on full-thickness skin defects after deep dermal burn. Burns 2007;33:642-8. [CrossRef]
20. 21. Ding SJ. Biodegradation behavior of chitosan/calcium phosphate composites. J Non-Cryst Solids 2007;353:2367-73. [CrossRef]
21. 22. Abdel-Fattah WI, Jiang T, El-Bassyouni Gel-T, Laurencin CT. Synthesis, characterization of chitosans and fabrication of sintered chitosan microsphere matrices for bone tissue engineering. Acta Biomater 2007;3:503-14. [CrossRef]
22. 23. Berscht PC, Nies B, Liebendörfer A, Kreuter J. Incorporation of basic fibroblast growth factor into methyl pyrrolidinone chitosan fleeces and determination of the in vitro release characteristics. Biomaterials 1994;15:593-600. [CrossRef]
23. 24. Wang YC, Lin MC, Wang DM, Hsieh HJ. Fabrication of a novel porous PGA-chitosan hybrid matrix for tissue engineering. Biomaterials 2003;24:1047-57. [CrossRef]
24. 25. Germershaus O, Mao S, Sitterberg J, Bakowsky U, Kissel T. Gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan block copolymers: establishment of structure-activity relationships in vitro. J Control Release 2008;125:145-54. [CrossRef]
25. 26. Shen JY, Pan XY, Lim CH, Chan-Park MB, Zhu X, Beuerman RW. Synthesis, characterization, and in vitro degradation of a biodegradable photo-cross-linked film from liquid poly(epsiloncaprolactone-co-lactide-co-glycolide) diacrylate. Biomacromolecules 2007;8:376-85. [CrossRef]
26. 27. Yao F, Chen W, Wang H, Liu H, Yao K, Sun P, Lin H. A study on cytocompatible poly(chitosan-g-L-lactic acid). Polymer 2003; 44:6435-41. [CrossRef]
27. 28. Mori T, Okumura M, Matsuura M, Ueno K, Tokura S, Okamoto Y, et al. Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro. Biomaterials 1997;18:947-51. [CrossRef]
28. 29. Norazril SAH, Aminuddin BS, Norhayati MM, Mazlyzam AL, Fauziah O, Ruszymah BH. Comparison of chitosan scaffold and chitosan-collagen scaffold: a preliminary study. Med J Malaysia 2004;59:186-7.
29. 30. Ishihara M, Ono K, Sato M, Nakanishi K, Saito Y, Yura H,et al. Acceleration of wound contraction and healing with a photocrosslinkable chitosan hydrogel. Wound Repair Regen 2001;9:513-21. [CrossRef]
30. 31. Ma J, Wang H, He B, Chen J. A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts. Biomaterials 2001;22:331-6. [CrossRef]
31. 32. Chuang WY, Young TH, Yao CH, Chiu WY. Properties of the poly (vinyl alcohol)/chitosan blend and its effect on the culture of fibroblast in vitro. Biomaterials 1999;20:1479-87. [CrossRef]