Several natural bioactive molecules have been used in the development of scaffolds to enhance biocompatibility or biodegradability and macroalgae contain many bioactive compounds that regulate the physiological activities of cells. In this study, extrapolymeric substances (EPS) from brown algae, Undaria pinnatifida, were dispersed in poly-ε-caprolactone (PCL) nanofiber, fabricated by electrospinning technique to mimic natural extracellular matrix (ECM), and tested as a scaffold for the production of artificial skin using rat primary fibroblasts. The level of adhesion, viability, and infiltration of cells on the EPS-PCL nanofibers were then assessed. The primary fibroblasts attached well, had good viability, and infiltrated through the nanofiber mat without cytotoxicity. Additionally, fibroblast on EPS-PCL nanofiber overcame the stress derived from high cell density at limited area. These results indicate that EPS-imbedded nanofiber has the potential to be used as scaffolds to develop artificial skin or as wound-healing nanomedicines to regenerate injured skin. 1. Introduction Skin protects the inner organs of an organism from a variety of stresses. Skin is primarily composed of three layers, the epidermis, dermis, and hyperdermis, each of which contains several types of cells. The dermis layer is made almost completely of fibroblasts, which are known to have an effect on keratinocyte proliferation in the epidermis and to be related to some immune responses of skin [1, 2]. When the skin is damaged at about 80？μm, the protected dermis layer can be exposed to air, necessitating regeneration to protect inner tissues. Moreover, in cases in which autoregeneration does not work well and serious scars are left, it is necessary for artificial skin to replace the injured skin for further medical treatments [3, 4]. Artificial skin can be made from skin cells cultured in/on three-dimensional biomaterials. Artificial skin requires a scaffold that is able to offer an environment that enables cells to grow well to establish a cellular community as well as to control their proliferation. Considering that all natural tissues make their complete functions via incorporation with extracellular matrix (ECM), scaffolds should be equivalent to ECM and provide cells with sufficient chances to attach to and spread along scaffolds and activate cellular metabolism normally [5]. To mimic ECM, microenvironment for activating cellular metabolism, nanofibrous scaffold has been intensively applied in tissue engineering. Each nanofiber strand supplies cell attachable point such as
References
[1]
T.-L. Tuan, L. C. Keller, D. Sun, M. E. Nimni, and D. Cheung, “Dermal fibroblasts activate keratinocyte outgrowth on collagen gels,” Journal of Cell Science, vol. 107, no. 8, pp. 2285–2289, 1994.
[2]
C. Bouffi, C. Bony, C. Jorgensen, and D. No？l, “Skin fibroblasts are potent suppressors of inflammation in experimental arthritis,” Annals of the Rheumatic Diseases, vol. 70, no. 9, pp. A24–A25, 2011.
[3]
T. Jaksic and J. F. Burke, “The use of “artificial skin” for burns,” Annual Review of Medicine, vol. 38, pp. 107–117, 1987.
[4]
G. Orlando, K. J. Wood, R. J. Stratta, J. J. Yoo, A. Atala, and S. Soker, “Regenerative medicine and organ transplantation: past, present, and future,” Transplantation, vol. 91, no. 12, pp. 1310–1317, 2011.
[5]
K. G. Cornwell, A. Landsman, and K. S. James, “Extracellular matrix biomaterials for soft tissue repair,” Clinics in Podiatric Medicine and Surgery, vol. 26, no. 4, pp. 507–523, 2009.
[6]
H. Hosseinkhani, Y. Hiraoka, C. H. Li et al., “Engineering three-dimensional collagen-IKVAV matrix to mimic neural microenvironment,” ACS Chemical Neuroscience, vol. 4, no. 8, pp. 1229–1235, 2013.
[7]
H. Hosseinkhani, M. Hosseinkhani, A. Khademhosseini, and H. Kobayashi, “Bone regeneration through controlled release of bone morphogenetic protein-2 from 3-D tissue engineered nano-scaffold,” Journal of Controlled Release, vol. 117, no. 3, pp. 380–386, 2007.
[8]
H. Hosseinkhani, M. Hosseinkhani, S. Hattori, R. Matsuoka, and N. Kawaguchi, “Micro and nano-scale in vitro 3D culture system for cardiac stem cells,” Journal of Biomedical Materials Research A, vol. 94, no. 1, pp. 1–8, 2010.
[9]
A. Subramanian, U. M. Krishnan, and S. Sethuraman, “Fabrication of uniaxially aligned 3D electrospun scaffolds for neural regeneration,” Biomedical Materials, vol. 6, no. 2, Article ID 025004, 2011.
[10]
W.-J. Li, C. T. Laurencin, E. J. Caterson, R. S. Tuan, and F. K. Ko, “Electrospun nanofibrous structure: a novel scaffold for tissue engineering,” Journal of Biomedical Materials Research, vol. 60, no. 4, pp. 613–621, 2002.
[11]
X. M. Mo, C. Y. Xu, M. Kotaki, and S. Ramakrishna, “Current research on electrospinning of silk fibroin and its blends with natural and synthetic biodegradable polymers,” Biomaterials, vol. 25, no. 10, pp. 1883–1890, 2004.
[12]
K. Senni, J. Pereira, F. Gueniche et al., “Marine polysaccharides: a source of bioactive molecules for cell therapy and tissue engineering,” Marine Drugs, vol. 9, no. 9, pp. 1664–1681, 2011.
[13]
K.-J. Kim and B.-Y. Lee, “Fucoidan from the sporophyll of Undaria pinnatifida suppresses adipocyte differentiation by inhibition of inflammation-related cytokines in 3T3-L1 cells,” Nutrition Research, vol. 32, no. 6, pp. 439–447, 2012.
[14]
L. Rittié and G. J. Fisher, “Isolation and culture of skin fibroblasts,” Methods in Molecular Medicine, vol. 117, pp. 83–98, 2005.
[15]
R. Djurhuus, A. M. Svardal, and E. Thorsen, “Toxicity of hyperoxia and high pressure on C3H/10T1/2 cells and effects on cellular glutathione,” Undersea and Hyperbaric Medicine, vol. 25, no. 1, pp. 33–41, 1998.
[16]
W. Konsavage, L. Zhang, T. Vary, and J. S. Shenberger, “Hyperoxia inhibits protein synthesis and increases eIF2α phosphorylation in the newborn rat lung,” American Journal of Physiology—Lung Cellular and Molecular Physiology, vol. 298, no. 5, pp. L678–L686, 2010.
[17]
F. Brugè, E. Venditti, L. Tiano, G. P. Littarru, and E. Damiani, “Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: is β-actin a reliable normalizer for photoaging studies?” Journal of Biotechnology, vol. 156, no. 3, pp. 153–162, 2011.
[18]
K. C. Kregel and H. J. Zhang, “An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations,” American Journal of Physiology—Regulatory Integrative and Comparative Physiology, vol. 292, no. 1, pp. R18–R36, 2007.
[19]
K. F. Ferri and G. Kroemer, “Organelle-specific initiation of cell death pathways,” Nature Cell Biology, vol. 3, no. 11, pp. E255–E263, 2001.
[20]
W.-X. Zong and C. B. Thompson, “Necrotic death as a cell fate,” Genes and Development, vol. 20, no. 1, pp. 1–15, 2006.
[21]
J. Han, S. Kang, R. Choue et al., “Free radical scavenging effect of Diospyros kaki, Laminaria japonica and Undaria pinnatifida,” Fitoterapia, vol. 73, no. 7-8, pp. 710–712, 2002.
[22]
N. Nishibori, T. Sagara, T. Hiroi, et al., “Protective effect of Undaria pinnatifida sporophyll extract on iron induced cytotoxicity and oxidative stress in PC12 neuronal cells,” Phytopharmacology, vol. 2, no. 2, pp. 271–284, 2012.
[23]
F. Tian, H. Hosseinkhani, M. Hosseinkhani et al., “Quantitative analysis of cell adhesion on aligned micro- and nanofibers,” Journal of Biomedical Materials Research A, vol. 84, no. 2, pp. 291–299, 2008.
[24]
N. C. Hunt, R. M. Shelton, and L. M. Grover, “Reversible mitotic and metabolic inhibition following the encapsulation of fibroblasts in alginate hydrogels,” Biomaterials, vol. 30, no. 32, pp. 6435–6443, 2009.
[25]
B. M. Baker, A. O. Gee, R. B. Metter et al., “The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers,” Biomaterials, vol. 29, no. 15, pp. 2348–2358, 2008.
[26]
J. B. Lee, S. I. Jeong, M. S. Bae et al., “Highly porous electrospun nanofibers enhanced by ultrasonication for improved cellular infiltration,” Tissue Engineering A, vol. 17, no. 21-22, pp. 2695–2702, 2011.
