# Fabricating a Novel Three-Dimensional Skin Model Using Silica Nonwoven Fabrics (SNF)
## 3 Results  
### Three-Dimensional Model of SNF Skin Using Immortalized Cells  
First, we compared the models using only epidermal cells by comparing Epidermal and SNF epidermal models. Morphological observations using hematoxylin-eosin staining showed that an epidermis-like layer was constructed on the cell culture insert membrane in the Epidermal model and SNF epidermal model, while some HaCaT cells were present inside the SNF. In addition, the SNF epidermal model (63.8 \(\pm\) 7.1 \(\upmu\)m) had a thicker epidermal layer than the Epidermal model (47.1 \(\pm\) 17.4 \(\upmu\)m) (Figure 2a,b,e). The cells of both models had the same round shape in most parts of the model. The SNF epidermal model also showed cells in the lower epidermal layer where SNF was present. For the RT-qPCR, human genes _hKRT10_ (Keratin 10), _hTGMI_ (Transglutaminase 1), _hTGM2_ (Transglutaminase 2), _hIVL_ (Involucrin), _hFLG_ (Filaggrin), _hLOR_ (Loricrin), and _hZNF750_ (Zinc finger protein 750) were selected as epidermal layer markers in this experiment. The mouse genes _mCol1a1_ (Collagen I xl), _mCol3_ (Collagen III), and _mCol4_ (Collagen IV) were selected as dermal layer markers. The expression of _hTGM2_ showed no significant difference in both models. The expression of _hKRT10_, _hTGMI_, _hIVL_, _hFLG_, _hLOR_, and _hZNF750_ was significantly upregulated in the Epidermal model compared with that in the SNF epidermal model (Figure 3).  
To examine the material of the dermal layer, we compared the SNF dermal/epidermal model with the Collagen dermal/epidermal model. Morphological observations using hematoxylin-eosin staining showed that the Collagen dermal/epidermal model (87.1 \(\pm\) 13.7 \(\upmu\)m) had a thicker epidermal layer than the SNF dermal/epidermal model (63.8 \(\pm\) 7.1 \(\upmu\)m) (Figure 2c-e). Furthermore, the cells in both models were round in the lower part and flattened in the upper part. In the Collagen dermal/epidermal model, the epidermal layer formed on top of the collagen and some fibroblasts could be seen in the dermal collagen layer. In contrast, in the SNF dermal/epidermal model, few cells were observed in the SNF, and the epidermal layer formed on top of the SNF. The results of RT-qPCR showed that the expression of _hTGM2_ and _mCol4_ was upregulated in the SNF dermal/epidermal model compared with the Collagen dermal/epidermal model, while the expression of _hKRT10_, _hIVL_, _hFLG_, _hLOR_, _hZNF750_, and _mCol3_ was significantly upregulated in the Collagen dermal/epidermal model compared with the SNF dermal/epidermal model (Figure 3).  
We also compared the SNF epidermal model and SNF dermal/epidermal model to determine the effect of fibroblasts in the SNF layer. Morphological observations using hematoxylin-eosin staining showed that there was no significant difference in epidermal thickness between the SNF epidermal model (63.8 \(\pm\) 7.1 \(\upmu\)m) and the SNF dermal/epidermal model (65.2 \(\pm\) 10.9 \(\upmu\)m) (Figure 2b,d,e). The results of RT-qPCR showed that the expression of _hTGM1_, _hIVL_, _hFLG_, and _hLOR_ was significantly upregulated in the SNF epidermal model compared with that in the SNF dermal/epidermal model (Figure 3).  
Figure 2: (**a–d**) Morphological observation of a skin models using immortalized cells. (**a**) Representative hematoxylin and eosin images of the Epidermal model, (**b**) SNF epidermal model, (**c**) Collagen dermal/epidermal model, and (**d**) SNF dermal/epidermal model. (**e**) Thickness of epidermal layer of each skin model constructed with immortalized human and mouse cells (\(n\) = 10, \(t\) \(p\) \(<\) 0.05, \(t\)\(t\)\(p\)\(<\) 0.01). Data is represented as the mean \(\pm\) standard error of the mean (sem); five picture \(t\)-test. Scale bar 50 \(\upmu\)m.