# In Situ Transformation of Electrospun Nanofibers into Nanofiber-Reinforced Hydrogels
## 4 Discussion  
In our study, we achieved a successful transformation of all electrospun samples from two-dimensional nanofibrous scaffolds into three-dimensional hydrogels upon contact with water (Figures 2 and A1). While the uniaxially electrospun fiber scaffolds (samples G and GPP) completely turned into hydrogels, hydrogels containing nanofibers (nanofiber-reinforced hydrogels) were formed from the coaxially electrospun core-shell scaffolds (samples zeinPEO-GPP and zeinPEOT-GPP) (Figure 2). The formation of nanofiber-reinforced hydrogels from the latter samples is associated with crosslinked gelatin swelling in water, while hydrophobic zein neither takes up much water nor dissolves in water, hence remaining intact, consistent with our previous work on zein fiber scaffolds and that of other studies [9; 34]. In contrast, transformation of the uniaxially electrospun gelatin nanofibers (samples G and GPP) was found to be associated with a higher water uptake, volume change and mass loss as compared to the coaxially electrospun nanofibers (zeinPEO-GPP and zeinPEOT-GPP) (Figure 3b). Since no zein was present in the uniaxial samples, the large water uptake during complete transformation of these samples into hydrogels with no stabilizing fibers present makes the hydrogel mechanically weaker, i.e., prone to tear, as has been described previously for hydrogel systems [2; 9; 54]. As described earlier, for  
Figure 7: Results for the cell viability studies. (**a**) MTT assay and (**b**) LDH assay for human dermal fibroblasts in the presence of different nanofiber scaffolds or aqueous PDA and PEI solutions in different concentrations, respectively. The data are presented as means with standard deviations of \(p\) < 0.0332 (*), \(p\) < 0.0021 (**) and \(p\) < 0.0001 (***). Key: G, fiber sample containing gelatin; GPP, fiber sample containing gelatin crosslinked by polydopamine and polyethyleneimine; zeinPEO-GPP, fiber sample containing zein and polyethylene oxide in the core and gelatin crosslinked by polydopamine and polyethyleneimine in the shell; zeinPEOT-GPP, fiber sample containing zein, polyethylene oxide and tetracycline hydrochloride in the core and gelatin crosslinked by polydopamine and polyethyleneimine in the shell; T, tetracycline hydrochloride control sample; PDA, polydopamine; PEI, polyethyleneimine. skin regeneration purposes, a high enough pore size is relevant for cell migration into the scaffold and skin regeneration. We found the highest pore sizes of \(\sim\)30 \(\upmu\)m for our nanofiber-reinforced hydrogels, which is beneficial for migration of fibroblasts (3-15 \(\upmu\)m in diameter [55]). As mentioned above, the mass loss of all scaffolds may be associated with incomplete crosslinking and potentially dissolution of gelatin and leaking of PDA and PEI from the fiber shell. In our study, the formation of PDA was induced by decomposition of (NH4)2 CO3 to release NH3 vapors, which act as an oxidant on dopamine HCl [41] and induce rearrangement of dopamine into different quinone structures, such as 5,6-dihydroxyindole, as well as the formation of self-assembled trimers of (dopamine)2/5,6-dihydroxyindole [56; 57]. With respect to interaction with PEI and gelatin, it has been described that after formation of PDA, PEI is covalently linked to the polymer [45] and that PDA and PEI crosslink with gelatin [38; 41]. Overall, however, it is unclear how efficient these crosslinking processes are as they are most likely determined by many factors, including the penetration depth of the NH3 vapors. It seems possible, that crosslinking in our study predominantly occurred on the surface and in a non-exhaustive fashion, which is in accordance with a previous study, which confirmed the presence of free PEI in electrospun samples [41]. With respect to the contact angle (wettability), surface properties, including microstructure and hydrophilicity of the fiber scaffolds are of importance.