# Electrospun Nanofibrous Membranes for Tissue Engineering and Cell Growth
## 4 Gelatin and Collagen
Crosslinking was found to significantly increase the biostability of the nanofiber mats, while chondroite was reported to significantly promote cell proliferation, as compared to pure collagen. This was explained by the role of chondroite sulfate, which induces skin tissue regeneration [62], osteoblast adhesion [63], and fibroblast proliferation [64]. Due to the chemical similarity of gelatin and collagen, gelatin can be expected to show similar effects in increasing cell adhesion and proliferation. [PERSON] et al. used genipin cross-linked gelatin nanofiber mats as the base to include decellularized rat brain extracellular matrix as an active agent, before mesenchymal stromal cells (MSCs) from rat bone marrow were seeded onto these scaffolds [65]. They found the MSCs grew not only on top of the substrate, but also in the inner volume of the decellularized samples. Seeding these cells on pure gelatin nanofiber mats and those with an additional decellularized rat brain extracellular matrix showed growth on both sides of the nanofiber mats in multilayered cultures, especially for the second tissue. Incorporation of a decellularized rat brain extracellular matrix into gelatin nanofiber mats, however, was found to trigger differentiation of MSCs towards neural/glial precursor cells. An interesting approach was chosen by [PERSON] et al., who blended gelatin with the conductive polymer polyaniline (PAni) to prepare conductive electrospun scaffolds [66]. With conductive scaffolds, cell attachment, proliferation, migration, and even differentiation can be stimulated [67]. PAni, on the other hand, is known to be biocompatible [68] and thus a good candidate to make a nanofiber mat conductive. [PERSON] et al. seeded rat cardiac myoblast cells on these electrospun nanofiber mats and found they supported cell attachment and proliferation with different amounts of PAni, as compared to glass and other substrates, but also in comparison with pure gelatin nanofiber mats [66]. They found the cell morphology depended on the substrate structure, with cells growing on fibers with larger diameters showing microfilament-rich pseudopodia attached to single fibers (Figure 1a-c), while cells on thinner fibers were more spread out and exhibited a smooth muscle-like morphology (Figure 1d), similar to cells grown on glass substrates (Figure 1e) [66]. This was attributed to the higher roughness of the fibrous substrates, which provided a larger surface area on which cells could attach, as compared to smooth glass substrates and tissue culture-treated polystyrene, which is commonly regarded as the gold-standard. Like collagen, gelatin can also be blended with water-resistant or slowly degrading polymers to reduce or avoid crosslinking. [PERSON] et al. report about PCL/gelatin blended nanofiber mats used for tissue engineering [69]. They seeded mouse fibroblast cells on nanofiber mats with different blend ratios and found significantly increased cell viability and cell proliferation for the blends as compared to pure PCL scaffolds. This was attributed to the presence of gelatin in these nanofiber mats, which is generally known to result in good cell adhesion and proliferation. Combinations of gelatin with PCL are thus often found in the literature [70; 71; 72]. There are, however, many other gelatin blends reported to be advantageous. Comparing PLGA and PLGA/gelatin blended nanofiber mats, [PERSON] et al. found increased cell viability on PLGA/gelatin blends compared to pure PLGA, and in addition they also found better MC3T3-E1 cell proliferation and guidance for aligned nanofibers than for randomly oriented ones [73]. They mentioned that avoiding crosslinking induced easier cell attachment to the scaffolds, as crosslinking decreased bioactivity and hydrophilicity of the nanofiber mats. [PERSON] et al. investigated polyvinyl alcohol (PVA)/gelatin nanofiber mats after physical crosslinking by methanol and reported osteoblasts to attach firmly on these scaffolds, facilitated by gelatin and the crosslinking process [74].