# Behavior of Primary Human Oral Keratinocytes Grown on Invisalign(r) SmartTrack(r) Material
## 4 Discussion
The major finding of our study is that Invisalign(r) aligners alter the balance between different inflammatory mediators. We found a decrease in TNF-\(\alpha\) and IL-6 expression in HOKs grown on the aligners. A similar trend could also be seen for ICAM-1. TNF-\(\alpha\) is produced by epithelial cells in response to infection, especially at the initial infection phase [35]. Dysregulation of IL-6 is associated with numerous oral diseases, including periodontal disease, oral cancer and lichen plants [36]. ICAM-1 is responsible for leukocyte adhesion and their transepithelialmigration [37]. In contrast, the expression of IL-1\(\beta\) and IL-8 increased after culturing HOKs on the aligners. Similarly to TNFa, IL-1\(\beta\) is also produced by oral epithelium upon exposure to the pathogens and plays an important role in promoting the inflammatory response. IL-8 is an important chemoattractant, and even a small amount of IL-8 stimulates leukocyte migration [37; 38]. Dysregulation of leukocyte migration through IL-8 degradation by periodontal pathogen _Porphyromonas gingivalis_ is an important factor of periodontal disease pathogenesis [39]. Translation of our data to the clinical situation is rather tricky because both pro- and anti-inflammatory effects of aligners on HOKs were observed. For example, an increased expression of IL-8 might enhance infiltration of leukocytes, promote the inflammatory reaction, and therefore represents a risk factor for oral health [40; 41]. Similarly, enhanced production of IL-1\(\beta\) could be considered as a potential risk factor for the aligner therapy. To some degree, these pro-inflammatory effects might be balanced by the anti-inflammatory action of aligners. However, taken together, alterations in the balance between different inflammatory mediators will impact the host-microbe homeostasis. In the case of accompanying inflammatory diseases such as gingivitis, an altered inflammatory response could lead to faster progress in disease development. Our experiments with HOKs show that inflammatory parameters are not affected as much as it was concluded from our previous study using Ca9-22 cells [19]. Our previous study found substantial increases of all inflammatory parameters under the same experimental conditions [19], indicating different molecular responses by different types of epithelial cells to SmartTrack(r) aligner material. Furthermore, we have mentioned that HOKs, grown on the inner surface, generally exhibit lower levels of pro-inflammatory mediators compared to those grown on the outer surface. This finding might also have some relevance because in the clinical situation, only the inner surface has contact with oral epithelium. The barrier function of oral epithelium is realized through the interconnections of keratinocytes by various transmembrane proteins [42]. This mechanical barrier is the first layer of defense against exogenous noxious agents and bacterial invasion [43]. Among transmembrane proteins, integrins play a pivotal role in keratinocytes for cell adhesion to the extracellular matrix and cell detachment [44]. There is a strong relation between trans-mucosal resistance and the number of intercellular junctions [29]. E-cadherin, a transmembrane protein, plays an important role in the maturation and formation of these intercellular junctions. Hence, to assess the potential effect of aligners' material on the cell barrier function, we have investigated the gene expression of E-cadherin, ITG-\(\alpha\)6, and ITG-\(\delta\)4. After two days of incubation, no significant effect on epithelial barrier function parameters could be detected compared to TCP. After seven days, only the expression of ITG-\(\alpha\)6 increased, whereas E-cadherin and ITG-\(\delta\)4 expression significantly decreased. This expression pattern in HOKs differs strongly from oral squamous carcinoma cells.