# Electrospun Nanofibrous Membranes for Tissue Engineering and Cell Growth
## 5 Chitosan and Chitin  
Chitin belongs to the highly abundant natural polymers and is mainly found in marine crustaceans, shrimps, and crabs. It is insoluble in common solvents. Chitosan, on the other hand, is an important derivative of chitin, obtained by partial deacetylation of chitin under alkaline conditions or by enzymatic hydrolysis and is soluble in acidic aqueous media [79]. Figure 1: Morphology of myoblast cells 20 h after seeding on: (**a**) gelatin nanofiber mat; (**b**) 15:85 PANi-gelatin blend nanofibers; (**c**) 30:70 PANi-gelatin blend nanofibers; (**d**) 45:55 PANi-gelatin blend nanofibers; and (**e**) glass matrices. Staining for nuclei-bisbenzimide and actin cytoskeleton-phalloidin. Reprinted from [66], with permission from Elsevier. In spite of the problems in dissolving chitin, it is regularly reported in the literature as a possible substrate material for tissue engineering [80], since it shows similar properties to glycosaminoglycan, which is part of the ECM [81]. [PERSON] et al. thus prepared electrospun chitin nanofiber mats [81]. To dissolve the chitin powder, it was first irradiated by Co\({}^{60}\)\(\gamma\)-irradiation and then dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) for 20 days. Electrospinning resulted in ultrafine nanofibers with an average diameter 163 nm. These nanofiber mats degraded in vitro faster than chitin microfibers, promoted cell attachment and spreading of normal human keratinocytes and fibroblasts better than the chitin microfibers, and after coating with type I collagen also promoted cellular response in all cells under investigation. The authors interpreted these findings as a higher functional activity of collagen-coated chitin nanofibers in terms of cell attachment and spreading for normal human keratinocytes as well as fibroblasts. In a similar manner, [PERSON] et al. prepared a chitin solution by \(\gamma\)-irradiation and dissolving the resulting chitin in HFIP [82]. In addition, they investigated deacetylation for transforming the original chitin matrix into a chitosan matrix. Most groups, however, combine chitin or its derivatives with other polymers. For example, [PERSON] et al. blended the water-soluble carboxymethyl chitin with PVA to prepare an aqueous electrospinning solution [83]. The resulting nanofiber mats were cross-linked by glutaraldehyde vapor before human mesenchymal stem cells (hMSCs) were seeded on them. The cells were found to attach and spread in the washed nanofiber mats, which supported cell adhesion and proliferation (Figure 2). [PERSON] et al. prepared chitosan/PVA nanofibers with chitin whiskers in different concentrations from succininic acid/water as solvent [84]. The resulting nanofiber mats were crosslinked by GA vapor, followed by mineralizing hydroxyapatite onto them by applying ten-fold concentrated simulated body fluid and seeding MC3T3-E1 osteoblast cells. They found an increased cell viability and proliferation due to the addition of the chitin whiskers, as compared to pure chitosan/PVA nanofiber mats. This effect was attributed to the clearly modified surface of the nanofiber mats, which showed a rough, uneven structure due to the hybridization with hydroxyapatite, and resulting in significantly improved cell viability and proliferation. Electrospun nanocomposites of chitin nanofibrils and PCL were prepared by [PERSON] et al. [85]. The chitin nanofibrils were dispersed in 2,2,2-trifluoroethanol (TFE) in which PCL could also be dissolved, allowing for spinning nanofibers with different chitin:PCL ratios, which showed strongly decreasing average diameters with increasing chitin content. For high chitin nanofibril contents, the water contact angle was significantly reduced. When seeding human dermal fibroblasts (hDF) on these nanofiber mats, the nanofiber mats with high chitin content, in particular, showed cell penetration and migration inside the scaffold; oppositely to pure PCL nanofiber mats, where the cells stayed on the surface.