# Behavior of Primary Human Oral Keratinocytes Grown on Invisalign(r) SmartTrack(r) Material
## 1 Introduction  
Clear aligners, a series of clear plastic appliances, represent an esthetic and removable alternative to conventional fixed orthodontic treatment. Reinforced by the patients' request for less visible orthodontic devices and a systematic promotion policy of aligner companies, an increased demand for clear aligner treatment took place in the last decade [1, 2, 3]. This led to an intensified development and application of this orthodontic treatment modality [4]. Constant improvements in clear aligner technology resulted in increased numbers and complexity of aligner treatments [5, 6]. Based on increasing ranges of aligner application, orthodontists and general dentists use aligner techniques to treat orthodontic patients [7, 8]. Taken together, clear aligner therapy has moved in the clinical and scientific spotlight of the past years. Align Technology is the most dominant aligner company in the orthodontic aligner market, and its product Invisalign(r) is with more than 9 million aligner treatments market leader ([[https://www.aligntech.com/about](https://www.aligntech.com/about)]([https://www.aligntech.com/about](https://www.aligntech.com/about)) accessed on 20 March 2021). SmartTrack(r), the latest Invisalign(r) material, consists of polyurethane and co-polyester. Invisalign(r) clear aligners are produced by a thermoforming process. Each aligner is produced based on a 3D printed dental cast, reflecting the actual clinical situation for the corresponding aligner [9]. SmartTrack(r) consists of a flat surface on the outside and a rough surface on the inside. Each aligner leads to a gradually improving clinical situation during treatment and should be worn for 22 h per day [10]. To act as effectively as possible, aligners should only be removed for oral hygiene and food intake and, thus, remain most of the time in direct contact with vital oral structures. Considering that an average orthodontic treatment takes multiple months, clear aligners could affect oral health [11]. Therefore, biocompatibility and material safety are pivotal aspects of long-lasting treatments [12]. To depict the effects of aligner material on oral cells, aligner eluates have been studied on multiple cells, including gingival fibroblasts, keratinocytes, and breast cancer cells. These studies reported a relatively weak cytotoxic effect of aligners under _in vitro_ conditions [13; 14; 15]. Further, a recent meta-analysis reported inconsistency in the current literature concerning safety considerations. Further clinical and laboratory studies were needed to shed led on the biological impact of this emerging orthodontic field [3]. Oral health is mainly depending on the integrity of the epithelium, a barrier of the body to its environment. In general, the oral epithelium is comprised of two main structures: the surface stratified squamous epithelium and its deeper basement membrane with the underlying connective tissue including nerves, lymphatic and blood vessels [16]. Gingiva is a tooth surrounding tissue that is set up by connective and epithelial tissues. Its epithelium is stratified squamous keratinized in the region of the gingiva and the papillae [17]. As outermost cell type in the keratinized part of the oral cavity, oral keratinocytes play a pivotal role in the oral defense system [18]. Since clear aligners are directly in contact with oral epithelial structures and no data existed on the behavior of oral cells directly grown on solid clear aligner specimens, we described in a previous _in vitro_ study the effects of SmartTrack(r) material on human oral epithelial cells directly grown on the material [19]. This study was performed with oral squamous carcinoma (Ca9-22) cells to give a first insight into the SmartTracks(r) cellular effects under direct contact conditions. We found no evidence of negative effects on oral squamous carcinoma cells. Although oral squamous carcinoma cells are often used as a model of the oral epithelium in _in vitro_ research, these cells do not entirely reflect all properties of oral epithelium.