# Fabricating a Novel Three-Dimensional Skin Model Using Silica Nonwoven Fabrics (SNF)
## 4 Discussion  
In this study, we created a three-dimensional model of the skin using SNF with immortalized cells and primary cultured cells. Both immortalized and primary cultured cells have several advantages. immortalized cells have a long lifespan and their quality is maintained at the genetic level, even at high passages [35]. However, when immortalized keratinocytes are compared with primary cultured keratinocytes, the epidermis is thinner and does not have full differentiation potential in terms of morphology [37]. In addition, immortalized fibroblasts show a weaker response to cytotoxicity and differ from primary cultured fibroblasts in gene expression analysis [38]. immortalized cell lines were used to take advantage of the small experimental error and long lifespan, whereas primary cultured cells have characteristics similar to those of human skin. Therefore, in this study, we used both the methods. First, to evaluate the effect of SNF on the epidermis, we compared Epidermal and SNF epidermal models. Compared to the Epidermal model, the SNF epidermal model was thicker in the immortalized cell models (Figure 2a,b,e). We then analyzed the expression of genes related to keratinization, basal epidermal formation, barrier function, proliferation, and dermis formation in both immortalized and primary cultured cell models. The expression of _hTGM2_--the formation factor of the epidermal basal layer [39]--was significantly upregulated in the SNF epidermal model compared with that in the Epidermal model in primary cultured cell models (Figure 3). UPEgulated expression of _hTGM2_ means that it has high functions as a skin model. _hTGM2_ contributes to epithelial barrier function by reducing ECM degradation and stabilizing ECM-cell interactions [40,41]. This leads to cell adhesion [42], and stabilization of extracellular structures such as basement membrane complexes [43]. _hTGM2_ expression was upregulated in the SNF epidermal model, suggesting that skin modeling in SNF enhances cell and ECM function and stabilizes basement membrane structure. In contrast, the expression of _hKRT10_, and _hTGM1_ a factor of keratinization [44,45]; _hIVL_, _hFLG_, and _hLOR_, factors of barrier function [44], and _hZNF750_, a factor of cell proliferation [46], was significantly upregulated in the Epidermal model. SNF did not affect the promotion of keratinization but functioned as a basement membrane structure. Next, to examine the scaffold materials of the dermis, we compared the SNF dermal/epidermal and Collagen dermal/epidermal models. Compared to the SNF dermal/epidermal model, the Collagen dermal/epidermal model had a larger thickness in the immortalized cell models (Figure 2c-e). In terms of cell shape in the epidermis, cellsin both models became flattened as they migrated upward, suggesting epidermal keratinization in both models. RT-qPCR showed that the gene expression of _hTGM2_ and _mCol4_, the formation factor of the epidermal basal layer [39] and the basement membrane [47], respectively, was significantly upregulated in the SNF dermal/epidermal model in the immortalized cell models (Figure 3). On the other hand, the expression of _hTGM2_, the formation factor of the epidermal basal layer [39]; _hCOL1A1_ and _hCOL3_, present in the entire dermis and upper dermis [48], respectively, and _hCOL4_, component of basement membrane [47], was significantly upregulated in the SNF dermal/epidermal model in primary cultured cell models (Figure 4). The results that genes expressed in the epidermal basal layer and dermis were significantly upregulated in the SNF dermal/epidermal model compared with the Collagen dermal/epidermal model suggested that while fibroblasts in the Collagen dermal/epidermal model were covered by collagen, fibroblasts in rigid SNF were highly proliferative. _hCOL1_, _hCOL3_, and _hCOL4_ are associated with skin elasticity [20; 48; 49; 50]. Therefore, upregulated expression of _hCOL1_, _hCOL3_, and _hCOL4_ in SNF dermal/epidermal model means that it has high functions as a dermal/epidermal skin model.