Cytotoxicity of two fluoride-releasing adhesive tapes.

4. Discussion

Recent studies in medicine have examined drug releasing technology and technologies for controlling and regulating drug diffusion rates and their rate of reaching target organs [18, 19, 20]. Various forms of mucoadhesion products have been developed to extend drug residence times. Based on a similar concept, fluoride adhesive films have been developed to control the fluoride release rate in films affixed to teeth [21].

Nanotechnology can create materials or devices with new properties and functions by manipulating ultrafine materials on the atomic or molecular level [22]. Nano-type fluoride shows greater response due to better solubility than macro-type fluoride [23]. For fluoride adhesive films, NaF was added through nanofiber spraying. Not only was this done to enhance the film’s performance as a medium, but also to adjust its viscosity, elasticity, strength, adhesive force and surface tension. Fluoride adhesive film supplemented with pullulan was designed to take advantage of its easy composition change. The bio-stability of these two types of film was tested. MTT assay was performed on HGF and PDL cells to test fluoride adhesive films’ bio-stability. HGF and PDL cells were selected because fluoride adhesive films may have contact with enamel, gingiva and periodontal ligaments since they are applied to maxillary and mandibular arch units [7]. Despite the small possibility of direct contact between the fluorine adhesive film and PDL, experiments were conducted with both HGF and PDL cells to ensure the maximum safety of the product [24, 25, 26]. PF-PVA showed higher cytotoxicity than F-PVA in both cells. In HGF, F-PVA was considered non-cytotoxic as cell viability was not reduced by more than 30% compared to control. PF-PVA showed higher cytotoxicity compared to previous studies despite the fact that it showed lower antibacterial activity, contrary to our assumptions.

MTT assay is a simple and relatively accurate method for assessing changes in cell proliferation and apoptosis, while enabling simultaneous measurement of large quantities [27]. However, to dissolve non-water-soluble formazan, organic solvents are needed. It is also possible to damage cells when removing the medium. Activities may differ depending on the physiological conditions of a single cell or cell type [28]. A major limitation of the MTT assay is that it can only assess cell viability binarily. Therefore, apoptosis cannot be clearly determined by this method alone [28, 29]. Moreover, genetic analysis was performed to overcome this problem, and to examine the causes of necroptosis and to observe the conditions of cells that come into contact with fluoride adhesive films in diverse manners [30].

RNA sequencing was performed on HGF and PDL cells applied to fluoride adhesive films to identify and analyze DEGs. All genetic analyses were conducted with the same parameters as previous genetic experiments used: fold value of 2, normalized data log value of ?4, and p-value of ? 0.05 [31, 32].

Genes should be analyzed and grouped by function instead of independently since RNA sequencing involves tens of thousands of genes. GO is often used for this purpose [33, 34, 35]. It is a major bioinformatic initiative to unify gene attributes representation across all species. Genes with similar functions could be grouped as GO.

Gene category chart was used to identify which genes in what type of GO showed relatively greater changes in expression (Fig. 2). Cell aging or apoptosis may be associated with many types of GO. We observed changes in gene expression associated with aging, apoptotic process, cell death, cell migration, DNA repair, immune response, neurogenesis and secretion GO in HGF and PDL cells with both F-PVA and PF-PVA. Among the genes of cell death GO, which is considered to have the highest intuitive association with cytotoxicity among GOs, we examined genes that showed significant changes in expression.

When PF-PVA was applied, expression of the GADD45A, GADD45B and PPP1R15A genes increased in HGF cells. DNA repair, cell arrest and apoptosis are regulated by the GADD45 A and B genes. The GADD family protein PPP1R15A is also involved in apoptosis as it induces growth arrest and DNA damage [36, 37].

The MLKL and NSG1 genes were significantly upregulated when F-PVA was applied to HGF cells, while DFFB, CASP9, CASP8 and MYD88 genes were downregulated. The MLKL gene plays an important role in tumor necrosis factor (TNF) induced necroptosis [38, 39]. DNA damage also induces apoptosis through the NSG1 gene [40, 41]. While an increase in the expression of these genes contributed to cell death, the expression of other genes decreased, including the DFFB gene that induces chromatin condensation during apoptosis [42]; the CASP8/9 genes, a caspase family member also involved in apoptosis [43]; and the MYD88 gene [44]. Such results showed differences from fluoride adhesive films supplemented with pullulan.

It was found that PDL cells showed significantly more cell death GO expression changes than HGF cells. PF-PVA showed significant expression changes in 61 genes. Forty-eight in F-PVA, showing fewer genes with expression changes than in PF-PVA.

A clear conclusion cannot be reached on cytotoxicity based only on the number of genes from the results of analyzing changes in expression of various genes. However, a greater number of genes associated with cell death GO showed changes in expression with fluoride adhesive film supplemented with pullulan than conventional F-PVA. As a consequence, cell viability was affected. MTT assay results indicate this (Fig. 1).

Comparing DEGs of cells in the three groups using Venn diagrams, only PDL cells had contra-expressed genes between fluoride adhesive films, compared to the control (n = 33). Based on such findings, PDL cells are susceptible to more diverse changes than HGF cells depending on the type of fluoride adhesive film used.

DEG analysis requires significant effort to understand the reasons behind gene expression changes. Unless target genes are clearly defined, such efforts are likely to cause errors. Therefore, it is more efficient to find and analyze key pathways of genes [31, 45, 46]. GSEA is a program for analyzing significant gene sets in control and experimental groups using microarray or RNA-seq data. GSEA analyzes only human and mouse genes and is based on gene sets in the molecular signature database (MsigDB) [47, 48, 49]. Among the various methods available for identifying key pathways, the hallmark pathway method was used in this study [31].

When PF-PVA was applied, GSEA results showed that TNFA_SIGNALING_VIA_NFKB is the key pathway in HGF cells. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) is a protein family involved in regulation of inflammatory response and immune system; necroptosis; cell proliferation; and differentiation of epithelial cells [50, 51]. TNF-? induces sustained inflammation in the body and is linked to various inflammatory diseases, including rheumatoid arthritis [52]. As with the cytological response observed when pullulan is used as a component of fluoride adhesive films, it could be viewed as a regulatory process of TNF-? that follows the NF-?B pathway. A possible mechanism by which PF-PVA could cause an inflammatory response in gingiva appears to involve influencing TNF-? through NF-?B in HGF cells. Based on this key pathway, the application of PF-PVA reduced cell viability by triggering an inflammatory response caused by the film.

As mentioned earlier, GSEA was performed to identify functional profiles of over-expressed genes in a large set and associations between genes in a set. From multiple genes, it was possible to examine each gene associated with the key pathway individually. On HGF cells, the NaF-PVA/Pullulan double layer adhesive film showed 15 genes involved in the key pathway (Fig. 5). Of these genes, LDLR gene deficiency increases plasma cholesterol levels and increased atherosclerosis risk. Increase in LDLR affects vascular inflammation [53]. The chemokine (C-X-C motif) ligand 3 (CXCL3) gene is a gene of inflammation called macrophage inflammatory protein-2-? [54]. It could be deduced that TNF-? function was enhanced by expression changes in these 15 genes.

Comparing conventional fluoride adhesive film with control, GSEA results on HGF cells revealed no hallmark key pathway. In other words, GSEA based on genetic testing produced results that supported MTT assay results.

GSEA results on PDL cells identified at least five key pathways from genes that showed changes in expression in PF-PVA, F-PVA and both groups. TNFA_SIGNALING_VIA_NFKB was the key pathway in all GSEAs. F-PVA also showed very high significance for this key pathway, but PF-PVA showed even higher significance.

To improve the adhesive force of conventional fluoride adhesive films, double layer adhesive films containing pullulan and PVA of different solubility properties were developed and showed higher cytotoxicity than conventional fluoride adhesive films. The DEG analysis results also indicate that the bio-stability of PDL cells is more vulnerable than HGF cells for all fluoride adhesive films. In children, fluoride adhesive films should only come into direct contact with the periodontal ligament in a few cases. However, several factors to consider when applying fluoride adhesive films to children: exposed roots due to abnormal mastication, gingival recession due to orthodontic treatment, exposed periodontal ligaments due to trauma, and significant alveolar bone resorption due to a systemic disease [55, 56]. Furthermore, the size of the fluoride adhesive film should be considered to minimize gingival contact and fabrication of customized fluoride adhesive films should enhance product safety.

Pullulan has structural flexibility with a uniform and unique linkage pattern of hydroxyl groups on glucopyranose rings. In contrast to other polysaccharides, it possesses distinct film-forming and fiber-forming properties [57]. Consequently, it is widely used in drug delivery, gene delivery, plasma expanders, tissue engineering, wound healing and oral care products [57, 58]. It is difficult to assume that the pullulan layer causes cytotoxicity based on its components and characteristics. It is believed that low cell viability would be difficult to verify by separate analyses of fluoride adhesive film and pullulan. Further studies on the interactions between pullulan and fluoride adhesive film are needed to determine the cause of such effects. Additionally, conducting further comparative studies on films with varying pullulan ratios could provide valuable insights and substantiate these findings.

It is true that pullulan can increase the adhesion of the film, but its disadvantages are evident from the increased cytotoxicity of the film in this experiment. Therefore, it is not recommended as a replacement for traditional F-PAV. Further studies on improving the application method of pullulan or materials that can replace pullulan are needed to improve fluoride adhesive films’ stability.

5. Conclusions

In conclusion, this study confirmed PF-PVA exhibited high cytotoxicity in MTT assay and gene analysis using HGF and PDL cells. These results suggest that PF-PVA has lower biocompatibility levels than F-PVA.

Full-text article online at https://www.jocpd.com/articles/10.22514/jocpd.2024.115/htm