# Behavior of Primary Human Oral Keratinocytes Grown on Invisalign(r) SmartTrack(r) Material
## 4 Discussion  
Most previous studies of aligner effects on oral cells used eluate settings [13; 14; 15]. In our recent study, we investigated for the first time the behavior of oral squamous carcinoma cells directly grown on SmartTrack(r) aligner material and found no adverse effect on these cells [19]. Oral squamous carcinoma cells represent a well-established _in vitro_ model for test settings of oral epithelial cells [22; 23; 24] but have their limitations compared to primary oral epithelial cells. Thus, oral squamous carcinoma cells are less susceptible to apoptosis than primary epithelial cells [25], and their expression of some functional properties, particularly E-cadherin, is altered during tumor transformation [26]. Therefore, it has to be proven if the investigated effects of Invisalign(r) clear aligner material can be also be seen in non-neoplastic oral epithelial cells. In the gingival papilla region, a tissue designed for peripheral body defense [27], clear orthodontic aligners are in close contact with gingival epithelium. Oral keratinocytes are the main cell type in the gingival epithelial tissue, a barrier that separates the body from its environment Thus, we investigated the effect of SmartTrack(r) aligner material on HOKs in this study and tested for proliferation/viability, morphology, and the expression of various proteins involved in the epithelial barrier function and local inflammatory response. An important observation of our study is that primary epithelial cells, similarly to oral squamous carcinoma cells [19], do not grow on aligner surfaces. This was proven by assessing the proliferation/viability of epithelial cells using the CCK-8 method. Similar to Ca9-22, no toxic effect of aligner surfaces on cell viability was observed in HOKs. Invisalign(r) surfaces and tissue culture plastic exhibited similar hydrophilicity as measured by contact angles [19]. Therefore, the fact that HOKs grew on Invisalign(r) surfaces exhibiting low proliferation and different attachment and behavior compared to TCP could be related mainly to the different compositions of these materials. It is reported that the growth of different cell types, including keratinocytes, decreases for polyurethane compared to TCP [28]. Our findings regarding proliferation/viability are in accordance with [PERSON] et al., who demonstrated decreased metabolic activity in immortalized human keratinocyte N/TERT-1 cell line using Invisalign(r) eluates in saline solution and artificial saliva [14]. Microscopic analysis after two and seven days showed no substantial differences after both time points. This can be seen in both bright field microscopy and scanning electron microscopy images. Furthermore, we observed that HOKs distributed evenly, and some cluster-like structures were observed rather rarely. In contrast, in our previous study, Ca9-22 cells tend to cluster formation, especially after seven days of culture [19]. This clustering effect could rather be explained accordingly by an intrinsic cell reaction than by a material-dependent cell behavior. Oral epithelium acts as a mechanical barrier for the infection and is involved in the immune response [29]. Therefore, we focused on the gene expression of some proteins involved in these two crucial functions. Live/dead staining did not reveal any substantial changes in the overall amount of dead cells on aligner material. It should be noted that PI, which is used in live/dead staining, recognizes only the cells in the late apoptosis. Additional staining with annexin V might be necessary to distinguish early apoptotic cells. However, because the number of PI\({}^{+}\) cells was very low, we do not expect any influence of apoptosis on gene expression results. Oral keratinocytes express a variety of cytokines and chemokines, such as IL-1\(\alpha\), IL-1\(\beta\), TNF-\(\alpha\), IL-6, and IL-8 [30; 31; 32]. Besides, these inflammatory mediators are also involved in the development of gingivitis [29], a commonly reported side effect of orthodontic aligner therapy [33; 34].