Preparation, properties, and cell attachment/growth behavior of chitosan/acellular derm matrix composite materials

Tao Lu, Rui xin Li, Yan Zhang, Yu xian Yan, Yong Guo, Jing Guan, Ji min Wu, Zhi hong LI, Bo Ning, Shu jie Huang, Xi zheng Zhang

.

DOI: 10.4236/jbnb.2011.22016                

Abstract

Composite membranes and sponge scaffolds consisting chitosan (CS) and acellular derm matrix (ADM) in six ratios were prepared by solvent evaporation technique and freeze-drying method, respectively. The composite materials were characterized by water contact angle measurement, scanning electron microscopy (SEM), water absorption and HaCat cells compatibility. The SEM result showed that CS/ADM three-dimensional (3D) micro-porous structures were successfully produced. The water absorption value of all scaffolds was over 18 times of its initial weight, which is high enough for skin regeneration scaffold, but there were no significant differences of water absorption ratio between deionized water and PBS solution for same scaffold (P > 0.05). HaCat cells were distributed uniformly on the surfaces of membrane 4-6, and an almost confluent monolayer was formed on membrane 6 on the fifth day, whereas cells maintained round and spherical in shape on the surface of membrane 1. The results showed that the cell compatibility of pure CS membrane needed to be improved, and addition of ADM realized this purpose. The results of compatibility of HaCat cells on scaffolds showed that the cell proliferated well on the scaffolds 3 and 4. In our study, the cell’s attachment and growth on the composite membranes was mainly determined by the content of the membrane, whereas the cell’s attachment and growth in the scaffolds was determined by both the content and structure of the scaffolds.

Keywords

Chitosan; Acellular derm matrix; Membrane; Scaffolds; HaCat cell compatibility

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	[1] K. Helena, L. Manfred and S. Horst, “Assessment of the Human Epidermis Model Skin Ethic RHE for in Vitro Skin Corrosion Testing of Chemicals According to New OECD TG 431,” Toxicology in Vitro, Vol. 20, 2006, pp. 547-559. doi:10.1016/j.tiv.2005.11.008
[2]	[2] J. Hoffmann, E. Heisler and S. Karpinski, “Epidermal- skin-test 1000 (EST-1000)—A new Reconstructed Epi-dermis for in Vitro Skin Corrosivity Testing,” Toxicology in Vitro, Vol. 19, 2005, pp. 925-929. doi:10.1016/j.tiv.2005.06.010
[3]	[3] Organisation for Economic Co-operation and Develop-ment, “Acute Dermal Irritation/Corrosion,” OECD Guideline for the testing of Chemicals, No. 404, Organi-sation for Economic Cooperation and Development, Paris, France, 2002.
[4]	[4] Organisation for Economic Co-operation and Develop-ment, “In Vitro Skin Corrosion: Human Skin Model Test,” OECD guideline for testing of chemicals, No. 431, Organisation for Economic Co-operation and Develop-ment, Paris, France, 2004.
[5]	[5] J. K. F. Suh and H. W. T. Matthew, “Application of Chi-tosan-Based Polysaccharide Biomaterials in Cartilage Tissue Engineering: A Review,” Biomaterials, Vol. 21, 2000, pp. 2589-2598. doi:10.1016/S0142-9612(00)00126-5
[6]	[6] E.B. Denkba? and M. Odaba?i, “Chitosan Microspheres and Sponges: Preparation and Characterization,” Journal of Applied Polymer Science, Vol. 76, 2000, pp. 1637- 1643.doi:10.1002/(SICI)1097-4628(20000613)76:11<1637::AID-APP4>3.0.CO;2-Q
[7]	[7] K. G. H. Desai and H. J. Park, “Preparation and Charac-terization of Drug-Loaded Chitosan–Tripolyphosphate Microspheres by Spray Drying,” Drug Development Re-search, Vol. 64, 2005, pp. 114-128. doi:10.1002/ddr.10416
[8]	[8] Iyabo Adekogbe and Amyl Ghanem, “Fabrication and Characterization of DTBP-Crosslinked Chitosan Scaf-folds for Skin Tissue Engineering,” Biomaterials, Vol.26, 2005, pp. 7241- 7250. doi:10.1016/j.biomaterials.2005.05.043
[9]	[9] P. J. Vandevord, H. W. T. Matthew, S. P. Desilva, l. Mayton, B. Wu and P. H. Wooley, “Evaluation of the Biocompatibility of a Chitosan Scaffold in Mice,” Jour-nal of Biomedical Materials Research, 2002, Vol. 59, pp. 585-90. doi:10.1002/jbm.1270
[10]	[10] L. Ma, C.Y. Gao, Z. Mao, J. Zhou, J. Shen and X. Hu, “Han C: Collagen/Chitosan Porous Scaffolds with Improved Bio-stability for Skin Tissue Engineering,” Biomaterials, Vol. 24, 2003, pp. 4833-4841. doi:10.1016/S0142-9612(03)00374-0
[11]	[11] A. N. L. Rocha, T. N. C. Dantas, J. L. C. Fonseca and M. R. Pereira, “Permeation of Drugs in Chitosan Mem-branes,” Journal of Applied Polymer Science, Vol. 84, 2002, pp. 44-49. doi:10.1002/app.10185
[12]	[12] H. Ueno, H. Yamada, I. Tanaka and N. Kaba, Matsuura M, Okumura M, Kadosawa T and T. Fujinaga, “Accelerating Effects of Chitosan for Healing at Early Phase of Experi-mental Open Wound in Dogs,” Biomaterials, Vol.20, 1999, pp. 1407-1414. doi:10.1016/S0142-9612(99)00046-0
[13]	[13] C. Xiao, S. Gao, H. Wang and L. Zhang, “Blend Films from Chitosan and Konjac Glucomannan Solutions,” Journal of Applied Polymer Science, Vol. 76, 2000, pp. 509-15. doi:10.1002/(SICI)1097-4628(20000425)76:4<509::AID-APP8>3.0.CO;2-2
[14]	[14] T. W. Chung, Y. F. Lu, S. S. Wang, Y. S. Lin and S. H. Chu, “Growth of Human Endothelial Cells on Photo-chemically Grafted Gly-Arg-Gly-Asp (GRGD) Chito-sans,” Biomaterials, Vol. 23, 2002, pp. 4803-4809. doi:10.1016/S0142-9612(02)00231-4
[15]	[15] Park Y. J., Lee M. Y., Lee J. Y., Seol Y. J., Chung C. P. and Lee S. J., “Controlled Release of Platelet-Derived Growthfactor-BB from Chondroitin Sulfate-Chitosan Sponge for Guided Bone Regeneration,” Journal of Con-trolled Release, Vol. 67, 2000, pp. 385-394. doi:10.1016/S0168-3659(00)00232-7
[16]	[16] T. Suzuki, Y. Mizushima, T. Umeda and R. Ohashi, “Further Biocompatibility Testing of Silica-Chitosan Complex Membrane in the Production of Tissue Plasmi-nogen Activator by Epithelial and Fibroblast Cells,” Jour-nal of Bioscience and Bioengineering, 1999, Vol. 88: pp. 194-199. doi:10.1016/S1389-1723(99)80201-1
[17]	[17] I. Yamaguchi, S. Itoh, M. Suzuki, M. Sakane, A. Osaka and J. Tanaka, “The Chitosan Prepared from Crab Tendon I: The Characterization and the Mechanical Properties,” Biomaterials, Vol. 24, 2003, pp. 2031-2036. doi:10.1016/S0142-9612(02)00633-6
[18]	[18] J. Y. Lee, S. H. Nam, S. Y. Im, Y. J. Park, Y. M. Lee, Y. J. Seol, C. P. Chung and S. J. Lee, “Enhanced Bone Forma-tion by Controlled Growth Factor Delivery from Chitosan- Based Biomaterials,” Journal of Controlled Release, Vol. 78, 2002, pp. 187-197. doi:10.1016/S0168-3659(01)00498-9
[19]	[19] S. F. Badylak, F. Ling and R. Record, “Hodde J. Vascu-larization of 3-Dimensional Scaffolds. Symposium on Tissue Engineering Science,” Aegean Conferences Series, 2002, 4: 63.
[20]	[20] P. Boukamp, R. T. Petrussevska, J. Hornung, A. Mark-ham and N.E. Fusenig, “Normal Keratinization in a Spon- taneously Immortalised Aneuploid Human Keratinocyte cell Line,” The Journal of Cell Biology, Vol. 106, 1988, pp. 761-771. doi:10.1083/jcb.106.3.761
[21]	[21] V. Sundararajan, Madihally and Howard W.T. Matthew. “Porous Chitosan Scaffolds for Tissue Engineering,” Bio-materials, Vol. 20, 1999, pp. 1133-1142.
[22]	[22] J. Ma, H. Wang, B. He and J. Chen, “A Preliminary in Vitro Study on the Fabrication and Tissue Engineering Ap-plications of a Novel Chitosan Bilayer Material as a Scaf-fold of Human Neofetal Dermal Fibroblasts,” Biomaterials, Vol. 22, 2001, pp. 331-336. doi:10.1016/S0142-9612(00)00188-5
[23]	[23] E. Behravesh, M. D. Timmer, J. J. Lemoine, M. A. K. Liebschner and A.G. Mikos, “Evaluation of the in Vitro Degradation of Macroporous Hydrogels Using Gravimeter, Confined Compression Testing and Microcomputed To-mography,” Biomacromolecules, Vol. 3, 2002, pp. 1263-1270. doi:10.1021/bm020067+
[24]	[24] J. R. Karel Smetana, Jaromir Lukas, Vera Paleckova and et al., “Effect of Ehemical Structure of Hydrogels on the Adhesion and Phenotypic Characteristics of Human Monocytes Such as Expression of Galectins and Other Carbohydrate-Binding Sites,” Biomaterials, Vol. 18,1997, pp. 1009-1014. doi:10.1016/S0142-9612(97)00037-9
[25]	[25] E. T. den Braber, J. E. de Ruijter, L. A. Ginsel and et al., “Quantitative Analysis of Fibroblast Morphology on Mi-crogrooved Surfaces with Various Groove and Ridge Dimensions,” Biomaterials, Vol. 17, 1996, pp. 2037-2044. doi:10.1016/0142-9612(96)00032-4
[26]	[26] R. Singhvi, G. Stephanopoulos and I. C. Daniel, “Effect of Substrate Morphology on Cell Physiology,” Biotech-nology and Bioengineering, Vol. 43, 1994, pp. 764-771. doi:10.1002/bit.260430811
[27]	[27] Takahiro Suzuki and Yasuyuki mizushima, “Characteris-tics of Silica-Chitosan Complex Membrane and Their Relationships to the Characteristics of Growth and Adhe-siveness of L-929 Cells Cultured on the Biomembrane,” Journal of Fermentation and Bioengineering, Vol.84, 1997, pp. 128-132. doi:10.1016/S0922-338X(97)82541-X
[28]	[28] N. J. Hallab, K. J. Bundy, K. O’Connor and et al., “Cell Adhesion to Biomaterials: Correlations between Surface Charge, Surface Roughness, Adsorbed Protein and Cell Morphology,” Journal of Long-Term Effects of Medical Implants, Vol. 5, 1995, pp. 209-231.
[29]	[29] T.G. Ruardy, H.E. Moorlag, J.M. Schakenraad and et al., “Growth of Fibroblasts and Endothelial Cells on Wet-tability Gradient Surfaces,” Journal of Colloid and Inter-face Science, Vol. 188, 1997, pp. 209-217. doi:10.1006/jcis.1997.4769
[30]	[30] C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides and D. E. Ingber, “Geometric Control of Cell Life and Death,” Science, Vol. 276, 1997, pp. 1425-1428. doi:10.1126/science.276.5317.1425
[31]	[31] E. A. Vogler and R. W. Bussian, “Short-Term Cell-At-tachment Rates: A Surface-Sensitive Test of Cell-Sub-strate Compatibility.” Journal of Biomedical Materials Research, Vol. 21, 1987, pp. 1197-1211. doi:10.1002/jbm.820211004
[32]	[32] Y. Zhu, C. Gao, T. He, X. Liu and J. Shen, “Layer-by- Layer Assembly to Modify Poly(L-Lactic Acid) Surface toward Improving Its Cytocompatibility to Human Endo-thelial Cells.” Biomacromolecules, Vol. 4, 2003, pp. 446- 452. doi:10.1021/bm025723k