Analysis of orthodontic aligner biocompatibility: leachable compounds of different aligner materials

Introduction

The development and use of aligners has become increasingly important in the orthodontic treatment, because of the growing desire of patients for appliances that are as inconspicuous and aesthetically pleasing as possible. The removable aligners need to be worn for around 22 h a day for 10–14 days. Depending on the malocclusion, the number of necessary aligners can be quite large. Consequently, the aligners are in contact with teeth, gums and intraoral fluids for a long period of time. Since the materials can be affected by the oral environment, molecule-release, that could have a harmful effect on oral cells, is possible [1]. In particular, one common synthetic substance that has been found to be an endocrine disruptor is bisphenol A (BPA). It has been connected to negative health side effects like obesity, cardiovascular disease, male and female infertility and developmental defects [2]. But BPA is more often being replaced by Bisphenol S (BPS), because it is considered to be safer due to its extremely decreased ability to leach out compounds. Leachables of BPS are associated with adverse effects on the cardiovascular system, the neural development and behaviour, the gut motility and the liver [3].

Previous studies have shown that even water is a plasticizer of polymer products, as it leads to a weakening of the intermolecular forces and consequently to chemical degradation. In addition, the degradation of polymers is accelerated by higher temperatures, mechanical wear and the presence of enzymes [4].

Although aligner therapy has become increasingly important in orthodontics, its systemic effects are largely unknown and there are only a few studies investigating the systemic toxicity of these materials. Since 2020, concerns about the toxicity of transparent aligners have increased. In vitro studies found that the resins used in clear aligners may have adverse effects on gingival cell activity, viability and reproductive capacity [5].

Conventional aligners are manufactured using the thermoforming process and are usually made of polyurethane with an integrated elastomer core. Recently, 3D-printed aligners have become increasingly popular due to technical developments. Each of these manufacturing techniques has advantages and disadvantages. Thermoformed aligners are associated with cytotoxicity, most likely due to the heating process required [6]. 3D-printed aligners made of polymethyl methacrylate (PMMA) are also considered as cytotoxic. The curing process might reduce, but does not completely eliminate this effect. Post-processing, such as polishing or sterilization (e.g. autoclaving or gamma irradiation) can also remove residual monomers [7].

A disadvantage of using synthetic polymers is the leaching of residual monomers into the saliva and possible consequent harmful biological reactions to the tissue [8]. Thavarajah et al. stated that the substances released from aligners could have a cumulative effect and thus lead to allergic, anaphylactic or non-specific reactions [9]. A similar situation was described by Tsuchiya et al. [10].

The incomplete conversion of monomers into polymers can increase the release of monomers, namely methyl methacrylate (MMA), triethylene glycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA) and bisphenol-A-glycidyl methacrylate (Bis-GMA) [11]. These monomers can cause local adverse effects such as cytotoxicity and mutagenicity as well as systemic effects such as teratogenicity and estrogenicity [12]. Rogers et al. reported severe reproductive toxicity after exposure of mouse oocytes to materials used in the manufacture of transparent aligners [5]. Martina et al. demonstrated a cytotoxic effect of various thermoplastic materials (Duran®, Biolon®, Zendura® and SmartTrack®) on human gingival fibroblasts in an in vitro experiment [13].

In contrast, Eliades et al. found no cytotoxicity of Invisalign brand aligners (Align™ Technology Switzerland GmbH, Switzerland) after they were stored in a saline solution in a glass container at 37 °C for two months [14].

The Tera Harz TC-85DAC (Graphy, Seoul, South Korea) has recently become available on the market. This is a material for printing transparent dental splints directly. According to the manufacturer, it is a biocompatible shape memory photopolymer. It has the CE (Conformité Européenne) as well as the KFDA (Korean Food and Drug Administration) certification for Class IIa medical devices and the FDA (Food and Drug Administration) 510 k approval. It is also GMP (Good Manufacturing Practice) and ISO13485 certified. However, as these aligners were recently launched on the market, there is only a little meaningful data available regarding the assessment of biocompatibility.

For this reason, the cytotoxicity effects of aligner materials should be intensively investigated. The aim of the work was to detect the determination of leachable compounds, including bisphenols, released from different aligner materials.

Materials and methods

Four different materials were selected for the analysis of their biocompatibility. The first one was a thermoforming splint CA® Pro Clear Aligner (Scheu Dental GmbH, Germany), which is a three-layer foil made of a copolyester double-shell construction and a thermoplastic elastomer core.

The second sample as another thermoforming splint Erkodur-al (Erkodent Erich Kopp GmbH, Germany), which is a copolyester (CAS no.: 261716-943).

The third product was also a thermoforming splint and is a SMP-Aligner, which was produced in a previous cooperation with the University of Leoben and consists of the components PPC (=polypropylene carbonate) and TPU (=thermoplastic polyurethane) with shape memory properties.

The last sample was a direct 3D printed aligner with shape memory properties made of the Tera Harz resin TC-85 DAC (Graphy Inc., South Korea) based on a vinyl ester urethane material.

For the analytical characterization three different analytical methods were used. The first one was a comparative characterization of the volatiles fraction based on headspace solid phase microextraction coupled to gas chromatography mass spectrometry (HS-SPME-GC-MS). Further on the two bisphenols A and S (BPA CAS-# 80-05-7 and BPS CAS-# 80-09-1) were quantified after extraction and derivatization by using gas chromatography tandem mass spectrometry (GC-MS). Finally, the transfer rate of leachable substances into artificial saliva under controlled conditions for various contact periods was analysed using HS-SPME-GC-MS.

Untargeted screening by HS-SPME-GC-MS

Samples of the different aligners were cut into small pieces of 100 mg and were weighed in a 20 mL headspace vial with a glass coated magnetic stir bar. The vials were sealed with a magnetic crimp cap with a PTFE (Polytetrafluorethylen) lined silicone septum. The volatile fraction was enriched on a 2 cm stable flex 50/30 µm DVB/Carboxen/PDMS (Supelco, Bellfonte, PA, USA) fiber for 20 min at 80 °C. The enriched volatiles were directly desorbed in the hot injection port of the GC-MS system at 270 °C (Gas chromatograph Shimadzu QP2010 with QP2020 single quadrupole mass selective detector, Shimadzu Duisburg, Germany and CTC PAL 1 liquid autosampler).

The separation was done a non-polar RXi5MS with 30 m 0.25 inner diameter and 1 µm film thickness (Restek, Bellfonte, PA, USA). The initial oven temperature was cooled down to −10 °C for optimum separation of the very volatile compounds.

Data acquisition was done in scan mode with a scan range from 35 - 350 amu (atomic mass unit) with a data acquisition rate of 3.3 scans per second.

For the identification two different mass spec libraries (NIST 23, FFSNC 4.0) were used. The initial value for identification was set to a similarity index (SI) of 80.

Determination of leachable compounds in artificial saliva

For the determination of organic compounds which can have migratable potential, 100 mg of each sample was suspended in 5 mL artificial saliva (Sialin – Sigma Saliva substitute batch No. 2731, Sigma-Pharm, Vienna, Austria) and stored in a closed vial in a climate cabinet for 24 h. After that time period the liquid was decanted from the sample material and replaced by freshly added 5 mL of saliva substitute and again stored for 24 h. This procedure was repeated for a third time under identical conditions for another 24 h. The three different saliva samples of 1 mL proportions were analyzed for volatiles using SPME under identical conditions.

For the quantification several deuterated compounds (toluene and n-alkanes) were added as internal standards in known concentrations.

Determination of bisphenols

Each sample, with a weight of 300 mg, was extracted in an ultrasonic bath in 2.5 mL methanol. Deuterated Bisphenol A-d16 was used an internal standard. The amount of 1 mL of the extract was reduced to dryness under a stream of nitrogen and derivatized to the corresponding silyl derivatives using MTBSTFA as derivatization agent and brought to a final volume of 1 mL with ethyl acetate as solvent.

Aliquots of 1 μL were injected into the gas chromatographic system with a tandem mass spectrometer as detector (Gas chromatograph Shimadzu QP2010 with TQ8050 tandem mass spectrometric detector and AOC 6000 autosampler, Shimadzu, Duisburg, Germany). Multiplier voltage was set to 1.6 kV and Q1 and Q2 were used in high resolution mode.

Results

Untargeted screening by HS-SPME-GC-MS

The four samples showed quite large differences in the qualitative and quantitative composition. The following chromatograms (Figures 1–4) display the four samples all in the same scaling for comparison of the results. While sample of the aligners CA® Pro Clear and Erkodur-al were almost free of detectable volatile compounds, the Graphy-Aligner showed the highest concentration and number of volatiles. The SMP-Aligner had one major compound which was identified as propylene carbonate (1,3-Dioxolan-2-one, 4-methyl-) and a second component which was quite likely to be 1,6-Dioxacyclododecane-7,12-dione (SI 88 %) or an adipate based plasticizer.

Figure 1:

Total ion chromatogram Graphy-Aligner.

Figure 2:

Total ion chromatogram SMP-Aligner.

Figure 3:

Total ion chromatogram CA® pro Clear-Aligner.

Figure 4:

Total ion chromatogram Erkodur-al-aligner.

The CA® Pro Clear-Aligner only emitted butylated hydroxytoluene (BHT), which is widely used as an antioxidant.

Table 1 describes the main components which were identified in the Graphy-Aligner.

Table 1:

The main components identified in the Graphy-Aligner.

Time, min	Area, %	Compound	SI
14.188	2.48	Cymene <para->	95
17.214	6.52	Isoborneol	97
17.356	3.36	Cyclohexanol, 5-methyl-2-(1-methylethyl)-	97
17.637	41.02	1,3-Dioxane-5-methanol, 5-ethyl-	93
19.658	5.11	Isobornylacetate	96
19.876	7.27	(3-Ethyloxetan-3-yl)methanol, acetate	86
19.979	10.72	Benzaldehyde, 2,4,5-trimethyl-	96
21.384	3.56	Isobornylpropionat	97
21.514	4.68	1,3-Dioxane-5-methanol, 5-derivative	84
21.699	5.1	Benzoic acid, 2,4,6-trimethyl-	96
23.404	3.92	Butylated hydroxytoluene	95
29.135	6.26	2-Ethenoxy-1,7,7-trimethylbicyclo(2.2.1)heptane	85

1. SI, similarity index; 100 would mean a perfect match from the acquired mass spectrometry and the data from the mass spec libraries. Most of the identified substances had a SI above 90, which indicates a good consistency of the measured mass spectrometry in comparison to the mass spectrometry in the used libraries. There are three compounds with a SI below 90, indicating that the proposed compound does not have a high-quality match, which raises some concern about the identity of the substance. If necessary, the substances need confirmation by analyzing pure reference compounds.

Figures 1–4 show the total ion chromatograms for the different types of aligners. The x-axis represents the time from the injection into the column until the sample is detected=retention time. The y-axis is defined as the measured response of the analyzed peak in the detector measured in milli-absorption units.

Determination of leachable compounds in artificial saliva

There were no leachable compounds detected in the samples of the aligners CA® Pro Clear and Erkodur-al. The identified substances in the samples of the Graphy- and SMP-Aligners are listed in the following Table 2.

Table 2:

Identified compounds in the samples of the Graphy- and SMP-Aligners.

	After 24 h	After 48 h	After 72 h	Total
Graphy-aligner	[µg/g]	[µg/g]	[µg/g]	[µg/g]
5,5-Dimethyl-1,3-dioxane	0.56	0.32	0.22	1.11
Norbornane <2,2-dimethyl-, 5-methylene->	0.35	0.29	0.21	0.85
Not identified	1.22	0.76	0.49	2.48
Bicyclo[2.2.1]heptan-2-ol, 1,5,5-trimethyl-	1.96	1.65	1.10	4.71
Bicyclo[2.2.1]heptan-2-ol, 2,3,3-trimethyl-	1.40	1.04	0.62	3.05
Isoborneol	8.25	7.42	5.33	21.00
1,3-Dioxane-5-methanol, 5-ethyl-	3.70	1.46	0.85	6.01
Not identified, probably a isobornyl-isomer	1.85	1.34	0.72	3.91
Isobornylacetate	3.09	4.19	3.41	10.69
1,3-Propanediol, 2-((acetyloxy)methyl)-2-ethyl-, diacetate	0.73	0.42	0.25	1.40
Benzaldehyde, 2,4,6-trimethyl-	7.01	6.41	5.16	18.58
Isobornylpropionate	0.65	0.63	0.48	1.77
Not identified, probably a 1,3-dioxane-5-methanol, derivative	0.62	0.38	0.22	1.22
SMP-Aligner	[µg/g]	[µg/g]	[µg/g]	[µg/g]
1,3-Dioxolan-2-one, 4-methyl-	0.29	0.17	0.12	0.58
2-Butoxyethyl acetate	0.26	0.14	0.10	0.50
Not identified, probably 1,6-dioxacyclododecane-7,12-dione or adipate	0.53	0.30	0.19	1.03

Figure 5 describes the release of leachable compounds from Table 2 as a percentage of total value.

Figure 5:

Release of leachable compounds from the graphy- and SMP-Aligner as a percentage of total value.

Discussion

In the work of Alhendi et al. who studied different thermoformed aligners, various chemical compounds could be detected by gas chromatography-mass spectrometry (GC-MS) [15]. Unknown compounds were identified by comparing the spectra with those of the NIST 2008 (National Institute of Standard and Technology Library) [16]. It was possible to identify benzene, 1,3-bis(1,1-dimethylethyl) as a chemical compound in all aligners at immersion solution concentrations of 100 and 75 % ethanol. They also found that alcohol concentrations below 50 % prevented the solution from degrading in the sample. Invisalign® proved to be the safest aligner of the systems tested – only one chemical compound (benzene) was confirmed. Eon® was classified as the least safe, as seven chemicals were detected in this system. Six and five detectable compounds were released from Clarity® and SureSmile® respectively. Although no traces of BPA were detected, the use of aligners is controversial regarding potential health concerns [15]. Schuster et al. also showed that there was no washout from Invisalign® aligners. It could be concluded that no residual monomers or oxidized by-products leached out in an immersion solution with 75 % ethanol [17]. Lower amounts of alcohol lead to hardly any release of chemical compounds [15]. A leaching process was also shown in this work. However, it must be added that ethanol was used in the studies of Alhendi et al. and Schuster et al. and artificial saliva in this current study. In this work, it was also possible to detect several chemical constituents and to identify them using mass spectra libraries. As aligners produced with different manufacturing processes were tested in this study, it could be shown, that there was a difference in terms of the leachable compounds between the materials.

Walele et al. tested aligner systems in an immersion solution of 75 % ethanol and were able to demonstrate the leaching of residual monomers [18]. Some of the detected compounds were classified as metabolites. For example, 4,6-dimethylundecane plays a biological role as a metabolite. 2,6,10,14-Tetramethylheptadecane and 3,5-dimethyloctane are metabolites found in human cancer metabolism [19]. The EU’s Globally Harmonized System (GHS) has classified nonadecane as a potentially hazardous aspiration hazard – if it enters the respiratory tract, this can have fatal consequences [19]. 1-Octadecane sulfonyl chloride was detected in SureSmile® at 100 % ethanol, whereas 3,5-bis(1,1-dimethylethyl) was present in both SureSmile® and Clarity® when tested at 50 % ethanol. These chemicals are classified by the GHS as a potential skin burn and irritation hazard. However, all potential side effects depend on several factors, such as incorporation, the concentration of the substance and the duration of contact. Phenol, 2,4-bis(1,1-dimethylethyl), is a bacterial metabolite as well as an antioxidant. It is an alkylbenzene and is classified by the GHS as hazardous to health and the environment. Depending on the absorption rate, it can cause organ toxicity, skin irritation and eye damage [20]. None of these leachable compounds were found in this study. While thermoformed aligners were almost free of leachable compounds, especially the printable one showed different amounts of various components. It is generally known that the toxicity of a biomaterial is directly proportional to the number of compounds released and their quantity [18].

In the clinical study by Raghavan et al., BPA levels were measured in the saliva of patients after placement of thermoformed retainers. The measurement took place before placement and after 1 day, 1 week and 1 month and showed statistically significantly increased values [21]. This coincides with the work of Azhagudurai et al. [22].

In contrast, Yazdi et al. showed in their systematic review of clinical and in vitro research on thermoplastic materials, no cytotoxic or estrogenic effects in clear aligner systems [23]. Furthermore, in other studies various thermoformed splints were tested as well for cytotoxicity in different solutions and concentrations and were found to be non-cytotoxic [13], 24], 25]. In addition, no proliferation of tumor cells of thermoformed aligners for use in orthodontics has been demonstrated in various studies [26], 27]. Iijima et al. also stated in their work that thermoformed aligners do not release any potential toxins that could cause adverse local or systemic reactions. They are not carcinogenic and do not cause developmental disorders [25].

Pratsinis et al. examined directly printed tooth repositioning splints fabricated with the resin Tera Harz TC-85 DAC (Graphy-Aligner) in sterile demineralized water. No signs of cytotoxicity on human gingival fibroblasts were detected. The antioxidant activity, expressed as the ability to reduce intracellular levels of reactive oxygen species, was not affected by the aligner samples. In addition, there was no significant induction of estrogenicity [28]. This is also in line with the study of Bleilöb et al. in which no cytotoxicity was found in printed aligners [29]. Recent in vitro studies also show that directly printed 3D aligners have a similar cytotoxic effect to thermoformed aligners in terms of fibroblast growth [30]. Willi et al. could not detect BPA in the directly printed aligners made of the photopolymerized resin Tera Harz [31]. These results are also consistent with the findings of Francisco et al. [32] and with the ones in this current study. However, it must be added that BPS could be detected in the sample of the Graphy-Aligner. Nevertheless, even in worst case scenario only 0.2 µg BPS from the aligner will be transferred to the human body. This is 250 times lower than the Specific Migration Limit (SML) of 0.05 mg/kg food and several orders of magnitude blow the recommended maximum of 20 mg/kg body weight per day [33].

Nevertheless, the production of aligners in large quantities has an impact on the ecosystem. Over time, plastic can degrade and lead to the formation of micro- or nanoparticles, resulting in the absorption of plastic additives by the environment. This means that humans and animals may be constantly exposed to these particles through their own food or water sources. This can lead to endocrine disruption and other problems related to plastic toxicity [34], 35].

It should be noted that the aging of aligners in the oral cavity does not correspond to the in vitro conditions, since, for example, the consumption of hot drinks can lead to high heat exposure. In addition, the oral environment could promote the release of BPA [14].

Although most studies report that the release of the monomers is below the toxic level, it should also be considered that most patients who use aligners are young and of a reproductive age.

The laboratory setting used in this study does not clearly reflect the environment in the oral cavity, as intraoral factors cannot be applied exactly in vitro. Aligners are exposed to mechanical abrasion, temperature fluctuations and enzymes during the wearing period [17].

Conclusions

No leaching of BPA could be detected in the thermoformed and the directly printed aligners. Nevertheless, it was evident that the 3D-printed aligner exhibited a higher number of leachable components compared to the thermoformed aligners.

However, it could be shown that leaching during the first 24 h of aligner usage was dominant compared to the other time intervals after 48 and 72 h. In addition, there was also a noticeable decrease in the released leachable compounds over the time span examined. Among the aligners tested, the Graphy-Aligner released the most substances, followed by the SMP-Aligner. CA Pro Clear® only leached Butylated Hydroxytoluene (BHT) and Erkodur-al no substances.

Due to the increased use of aligners, clinical studies are increasingly needed. To avoid possible cumulative negative effects, orthodontists should try to reduce the number of aligners used and the duration of treatment.