# Fabricating a Novel Three-Dimensional Skin Model Using Silica Nonwoven Fabrics (SNF)
## 4 Discussion
Fibroblasts secrete soluble factors that diffuse into the epidermal cells of the epithelium and induce the production of basement membrane proteins [51]. Therefore, SNF enhanced the function of fibroblasts and promoted a significant upregulation of basement membrane constitutive factors. These results suggest that the absence of fibroblasts in SNF was not because they were not present, but because the material may have been physically detached during the staining process (Figure 2). In contrast, factors related to keratinization and barrier function were more highly expressed in the Collagen dermal/epidermal model than in the SNF dermal/epidermal model. In a collagen skin model, epidermal growth was enhanced when the collagen matrix was organized by fibroblasts [10]. Therefore, in the SNF dermal/epidermal model, keratinization was observed at the visual level but inferior to the Collagen dermal/epidermal model at the genetic level. To examine the effect of fibroblasts in the SNF layer, we compared the SNF epidermal and SNF dermal/epidermal models. Morphological observations showed that there was no significant difference in epidermal thickness between the SNF epidermal model and the SNF dermal/epidermal model in the immortalized cell models, suggesting that fibroblasts do not affect epidermal thickness (Figure 2b,d,e). The models made from immortalized cells showed that _hTGM1_, factor of keratinization [45], _hVVL_, _hFLG_, and _hLOR_, factors of epidermal barrier function [44] were significantly upregulated in the SNF epidermal model compared with those in the SNF dermal/epidermal model (Figure 3). On the other hand, in primary cultured cell models, the gene expression of _hTGM1_, factor of keratinization [45] and _hTGM2_, factor of epidermal basal layer forming [39] was upregulated in the SNF dermal/epidermal model compared with the SNF epidermal model. The gene expression of the barrier function [44], _hVVL_ and _hFLG_ was significantly upregulated in the SNF epidermal model in primary cultured cell models. Barrier function was inferior when the model was cultured with fibroblasts in SNF. The expression of cornified envelope-associated genes related to barrier function, such as _hVVL_, _hFLG_, and _hLOR_ in HaCaT is different from that in NHEK [52]. From here, the difference in gene expression of these barrier function-related genes is due to cellular differences. Also, for the expression of _hTGM2_, there were no significant differences between immortalized and primary cultured cells, allowing for alternative gene expression analysis [52]. Therefore, the addition of fibroblasts to the SNF dermal layer takes advantage of SNF's basement membrane-mimicking structure. Furthermore, the usefulness of the SNF skin model as a three-dimensional skin model was evaluated by examining the skin corrosivity test using the MTT assay. Except for the Epidermal model, the other models showed skin corrosiveness theoretically, indicating that the MTT test could be performed. So, we demonstrated that skin corrosion tests are applicable on SNF [36]. Finally, we attempted to create a model that incorporated the advantages of both SNF and collagen. SEM results of the combination models showed that the interface between SNF and collagen adheres without repelling (Figure 6c-h). In this study, collagen was immobilized via physical adsorption, which is a common approach to glass surface modification, so the interface between SNF and collagen is firmly adhered due to van der Waals forces [53; 54]. The sCS model had collagen modifications in proportion to collagen concentration (Figure 6c,d). In the gCS model, although collagen accumulated in the upper part of the SNF due to the high viscosity of the collagen gel, the collagen gel-modified SNF was successfully formed (Figure 6e-h). Next, we examined the model using hematoxylin-eosin staining (Figure 7). For the SNF dermal/epidermal model and the Collagen dermal/epidermal model, the cells flattened as they approached the surface, indicating that the epidermal layer had become keratinized.