# Fabricating a Novel Three-Dimensional Skin Model Using Silica Nonwoven Fabrics (SNF)
## 3 Results
### Three-Dimensional Model of SNF and COLAGEN Using immortalized Cells  
In the combination model of SNF and collagen, we examined two models: the sCS (solution of collagen-treated SNF) model where the SNF was immersed in a collagen solution and the gCS (gel of collagen with SNF) model where the collagen gel was poured into the SNF. Each model was examined using different collagen concentrations. We first observed each model using SEM. The SNF dermal/epidermal model showed SNF lined up in a fibrous pattern with voids (Figure 6a). In the Collagen dermal/epidermal model, collagen covered the entire surface (Figure 6b). In the sCS models, collagen modification around the SNF was observed at both concentrations (Figure 6c,d). For the gCS models, both concentrations showed a high density of collagen modification on the SNF in the upper layer but voids were observed (Figure 6e,g). In the middle layer of the gCS model, SNF was modified with collagen like in the sCS model (Figure 6c-h). Figure 5: Results of MTT assay of the skin models using primary cells. Viability of cells in skin models after exposure to corrosive and noncorrosive substances, according to 431 OECD [35]. Dashed lines indicate 15% and 50% viability thresholds (\(n=2\)). Figure 6: (**a**–**h**) Scanning electron microscope (SEM) images of skin models. (**a**) SNF dermal/epidermal model, (**b**) Collagen dermal/epidermal model, (**c**) 0.01 sCS model, (**d**) 0.1 sCS model, (**e**) 1.2 gCS model top layer, (**f**) 1.2 gCS model medium layer, (**g**) 2.4 gCS model top layer, and (**h**) 2.4 gCS model medium layer. Scale bar 50 \(\upmu\)m.  
We compared these combination models with the SNF dermal/epidermal model and Collagen dermal/epidermal model. First, we compared them with a SNF dermal/epidermal model. Morphological observations using hematoxylin-eosin staining showed that there was no significant difference between the sCS model (0.01 sCS model: 70.9 \(\pm\) 9.6 \(\upmu\)m, 0.1 sCS model: 75.1 \(\pm\) 10.9 \(\upmu\)m) the SNF dermal/epidermal model (78.6 \(\pm\) 5.9 \(\upmu\)m) in epidermal thickness (Figure 7a,c,d,g). Also, both 1.2 gCS model (111.7 \(\pm\) 5.7 \(\upmu\)m) and 2.4 gCS model (128.0 \(\pm\) 9.7 \(\upmu\)m) had a thicker epidermal layer than the SNF dermal/epidermal model (78.6 \(\pm\) 5.9 \(\upmu\)m) (Figure 7b,e-g). Furthermore, the cells in the 1.2 and 2.4 gCS model were round in the lower part and flattened in the upper part, while the 0.01 and 0.1 sCS model had the same shape cells in most parts. In addition, in the 0.01 sCS model, most of the epidermal layer was formed on top of the SNF, although some cells were present in the SNF. On the other hand, in the 0.1 sCS, 1.2 gCS, and 2.4 gCS model, there were few cells in the SNF and the epidermal layer was formed on top of the SNF. The results of RT-qPCR showed that the expression of _hTGM1_, _hTGM2_, and _hLVL_ were significantly up-regulated in the 0.1 sCS model compared with the SNF dermal/epidermal model (Figure 8). _hLVL_, _hFLG_, _hLOR_, and _hZNF750_ were significantly upregulated in the 1.2 gCS model compared with the SNF dermal/epidermal model, while _mCol4_ was significantly upregulated in the SNF dermal/epidermal model (Figure 8). _hKRT10_, _hTGM1_, _hTGM2_, _hLOR_, _hLVL_, _hFLG_, and _hZNF750_ were significantly upregulated in the 2.4 gCS model compared with the SNF dermal/epidermal model, while _mCol4_ was significantly upregulated in the SNF dermal/epidermal model (Figure 8). Figure 7: (**a–f**) Morphological observation of skin models combining SNF and collagen. (**a**) Representative hematoxylin and eosin images of the SNF dermal/epidermal models, (**b**) Collagen dermal/epidermal model, (**c**) 0.01 sCS model, (**d**) 0.1 sCS model, (**e**) 1.2 gCS model, and (**f**) 2.4 gCS model. (**g**) Thickness of epidermal layer of each skin model constructed with immortalized human and mouse cells (\(n=10\), ++ \(p<0.01\), ++ \(p<0.001\)). Means \(\pm\) sem; five picture \(t\)-test.