Recent trends in contemporary orthodontics emphasize the growing adoption of clear aligner therapy (CAT) [1]. Significant scientific efforts have been focused on improving the materials and fabrication processes for CAT. Traditionally, clear aligners have been fabricated using a vacuum-forming or thermoforming process with amorphous polymer sheets and dental casts. However, with rapid advancements in three-dimensional (3D) technology and biomaterials, computer-aided design (CAD) and manufacturing (CAM) have been integrated into CAT, enabling the production of directly-printed aligners (DPA) [2]. In recent years, DPAs have garnered significant attention due to their environmental advantages and the customization potential of in-house fabrication systems [3], [4].

Although clear aligners are widely considered safe appliances with good biocompatibility, the dramatic increase in their use has led to reports of adverse clinical events [5], [6]. Allareddy et al. [5] documented 169 adverse events related to Invisalign use over a 10-year period starting in 2006, based on data from the Manufacturer and User Facility Device Experience (MAUDE) database. These events ranged from mild symptoms, such as rashes, to serious or life-threatening reactions, including difficulty breathing, sore throat, swelling of the throat, tongue or lips, hives, anaphylaxis, and sensations of airway obstruction or laryngospasm.

Only a limited number of studies have investigated the cytotoxic effects of clear aligners, with most focusing on the cytotoxic properties of thermoplastic materials [7], [8], [9], [10], [11]. These properties are influenced by multiple factors, including polymer composition and structure, fabrication processes, and environmental conditions such as temperature, humidity, pressure, and thermal history. Existing in-vitro studies have demonstrated that thermoformed aligners, typically made from glycol-modified polyethylene terephthalate and polyurethane, exhibit mild to moderate levels of cytotoxicity [7], [8], [9], [10]. Additionally, the thermoforming process has been shown to increase the release of monomers, thereby elevating cytotoxicity levels [9], [11].

In contrast, relatively few studies have examined the cytotoxicity and monomer release specifically from DPAs [12], [13], [14]. Since DPAs are often fabricated using in-house systems, concerns have been raised about the potential leaching of unpolymerized monomers and their toxic effects on biological systems [12], [13]. Willi et al. [13] detected urethane dimethacrylate (UDMA) in all tested samples of DPAs, with concentrations ranging from 29 to 96 μg/L after immersion in distilled water for one week, raising concerns about long-term intraoral exposure. Meanwhile, Pratsinis et al.[12] reported no cytotoxicity or change in intracellular reactive oxygen species (ROS) levels when human gingival fibroblasts were exposed to 20 % (v/v) eluates from DPAs. More recently, efforts have been made to optimize post-processing parameters for improved biocompatibility [14], [15], [16]. Studies by Iodice et al. [15] and Bleilöb et al. [16] investigated the influence of UV curing time and aligner thickness on cytotoxicity, confirming that a standard 20-minute UV curing protocol ensures acceptable biocompatibility, with longer curing times offering no additional benefit. These findings collectively highlight the importance of processing conditions in determining DPA safety, yet they also underline the lack of standardization across manufacturers and clinical settings.

A key limitation of in-vitro cytotoxicity testing is that it typically involves a one-time experimental setting [7], [8], [9], [10], [11], [12]. In CAT, however, patients are instructed to replace DPAs, approximately every 7–10 days [17], [18]. Clinically, adverse events are rarely associated with the first set of aligners, but are more commonly observed after 3–4 sequential replacements. This frequent replacement may contribute to cumulative exposure to leachable chemical substances, potentially compromising patient safety [13], [19]. Given these concerns, this study aims to evaluate the cytotoxicity of DPAs by simulating the sequential replacement of multiple DPAs in an in-vitro model. The null hypothesis is that sequential elution of DPAs under simulated oral conditions does not result in a significant increase in cytotoxicity. In addition, this study aims to identify potential leachable components from DPAs and assess post-processing methods that may reduce their cytotoxicity.