The introduction of transparent thermoplastic materials in orthodontics leads to increase in demand for more esthetically appliances not only for adults ¹ but for preadolescents and children. ² Patients, who prefer “invisible” treatments, have a tendency toward using clear aligner therapy instead of traditional metal brackets ³ and using transparent vacuum-formed retainers instead of Hawley-type retainers. ⁴

Clear aligners are plastic-based trays that could correct some dentofacial malocclusions in specific sequences. They must be worn approximately full-time (22 h a day) except during eating and oral cleaning procedures for 2 weeks and be replaced with another one until completing the duration of orthodontic treatment. ⁵ During the treatment period, aligners are in close contact with the oral environment for a long time and are continually exposed to heat changes, moisture, respiration, bacteria, and salivary enzymes, ⁶ and also to trauma in oral function like abrasion at the cusp tips or nonfunctional habits like bruxism. ⁷

Polyurethane is the basic component used in plastic-based materials that could be affected by changes in the oral environment surrounding plastics. ⁸ This contact leads to the release of biologically active substances that could be cytotoxic or estrogenic molecules which are able to cause biological reactions and modify gene expression. ⁹

One of the materials with estrogenic properties is bisphenol-A (BPA). This compound is starting monomer for the production of epoxy resins and polycarbonates which is found in the structure of clear aligners and retainers. ¹⁰ , ¹¹ , ¹² Intraorally, degradation of these materials could cause leach BPA due to changes in temperature or PH, mechanical wear, and bacterial or salivary enzymatic activity. ¹³ , ¹⁴ , ¹⁵ Estrogenic interactions are not only potentially harmful effect of BPA but also proliferative stimulation on human cells and toxicity activity were reported. ¹⁶ , ¹⁷ , ¹⁸ According to the United State Environmental Protection Agency (EPA) referenced the Food and Drug Administration protocol, safe intake dosage of BPA is 50 m kg/kg/day. ¹⁹ However, adverse events have been seen and mentioned with dosages even below this safe level. ²⁰

Few controversial studies have investigated the toxicity of clear aligners and their extent of BPA leaching. Some of them did not show any estrogenic or cytotoxic effects in treatments. ¹⁵ , ²¹ In contrast some others reported undesirable events when using aligners. ²² Therefore, the literature is inconsistent in this regard. ²³

Due to the above-mentioned controversies and lack of any systematic reviews in this regard, the aim of this systematic review was to summarize the biocompatibility and safety of clear aligners and transparent vacuum-formed retainers, such as their BPA release, adverse effects, cytotoxicity, and estrogenic effects.

This systematic review was prepared according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines.

Search strategy

Internet databases and search engines as well as reference lists of relevant articles were searched up until December 22, 2021, to find studies relevant to the biocompatibility of clear aligners and thermoplastic retainers. The Internet databases and search engines in use were Web of Science, PubMed, Cochrane, Scopus, and Google Scholar. The search keywords were related to the biocompatibility of clear aligners and thermoplastic retainers Table 1. The search was done by 3 researchers independently.{Table 1}

Population, intervention, comparator, and outcomes

The population, intervention, comparator, and outcomes items were as follows: The population was any studies (in vivo or in vitro). The intervention was the use of orthodontic clear aligners and transparent vacuum-formed thermoplastic retainers. The comparator was any control group compared with these experimental groups. The outcomes were any BPA release, adverse effects, cytotoxicity, and estrogenic effects of orthodontic clear aligners and transparent vacuum-formed thermoplastic retainers.

Eligibility criteria

The articles published in the literature were screened for the inclusion and exclusion criteria by three authors. This was first done independently, and after that, jointly through discussion. As for eligibility criteria, there was no year limit. Furthermore, articles in all languages would be included as long as their text could be translated clearly (using online translators or by professional translators). All types of publications (article, book, and thesis) would be included if containing relevant studies and information: The included publications should contain research methodology and results, and hence articles such as editorials or letter to editors or such that had only recommendations would be excluded. The included studies should have been on clear liners or thermoplastic retainers. In addition, they should have been on biocompatibility, safety, cytotoxicity, or estrogenicity of clear aligners or thermoplastic retainers. There were no restrictions on the type of study (e.g., randomized clinical trials and experimental in vitro studies). Studies focusing merely on the mechanical properties of clear aligners or thermoplastic retainers (without examining their chemical properties) would be excluded. Reference lists of the excluded studies were read and checked for relevant articles (e.g., references of articles related to the biocompatibility of other types of appliances other than clear aligners or thermoplastic retainers that had been excluded).

Data collection

The data were collected by a minimum of 3 researchers (and in some cases, 4 researchers), first independently and later through discussion. The outcomes of interest were cytotoxicity (cell viability, cell proliferation, cell reaction such as cell mobility and cell inflammatory response and barrier function), estrogenicity, or monomer and BPA leaching. The other variables sought were study design, sample size, technical analysis method, and grouping of interventions.

Risk of bias

Since there is no standard risk-of-bias assessment tool for in vitro studies, the authors borrowed items from other risk-of-bias assessment tools. A rather comprehensive list of items was assembled for investigating the risk of bias, by taking items from relevant renowned risk-of-bias tools for study types other than in vitro studies (since no risk-of-bias assessment tool existed for in vitro studies). All the included studies were screened for risk of bias using a minimum of 3 researchers (and in some cases, 4 researchers), first independently and afterward by discussion.

Data abstraction and synthesis

A panel of three authors examined the studies summarized as tables to be eligible for synthesis. All the units of measurement were converted to the same unit of ppm. There were no missing summary statistics. Sample heterogeneity was assessed using I ²statistics. Possible causes of heterogeneity among study results were assessed using subgroup analysis. Sensitivity analyses were carried out to assess the robustness of the synthesized results.

Statistical analyses

Only four studies had reported the leach of BPA. Since the methodological characteristics of these studies differed greatly and their timings were not matched, it was not possible to aggregate their results into a meta-analysis. Therefore, the only analysis performed was summarization: Their results were summarized and their variances were calculated.

Study characteristics

The initial search yielded 59 relevant articles. According to the selection criteria and after removing duplicates 19 articles were chosen. After removing 3 overlapped studies, 16 papers were found to meet the criterion, among which there were 15 in vitro investigations and 1 randomized control trial Figure 1and Table 2. Figure 1

PRISMA flow diagram showing the included studies. PRISMA: Preferred reporting items for systematic reviews and meta-analyses.

The increasing demand for the application of thermoplastic clear aligners in both active orthodontic treatment and retention phase indicates the necessity of a comprehensive assessment of this popular and new treatment approach. Fluctuations in oral cavity PH, humidity, pressure, and temperature in addition to the enzymatic function of bacteria and saliva lead to mechanical and chemical transformation of these thermoplastic materials which may result in leaching of unreacted components including BPA. ¹⁶ , ⁴⁴ , ⁴⁵ , ⁴⁶ , ⁴⁷ Ryokawa et al. evaluated aligners in a stimulated intraoral environment and revealed that these materials absorbed the highest amount of water after 24 and 336 h among all tested materials; and during the study, they did not saturate completely which showed the probability of monomer leaching from the aligners. ⁴⁸ BPA has been supposed to have a number of estrogenic or cytotoxic effects including disruption of beta-pancreatic cells' physiologic activity (insulin tolerance) and increased risk of prostate cancer and breast cancer. ⁴⁹ , ⁵⁰ Several publications confirmed adverse effects of BPA even at doses lower than the safe standard dose of 50 μg/Kg weight. ²⁰ , ⁵¹ , ⁵² , ⁵³ Patients usually use each aligner for 22 h every day approximately for 2 weeks; aligners wrap around the teeth and contact approximately a third of the gingiva; hence, their safety toward oral cavity cells including keratinocytes and fibroblasts is of great concern. A number of studies have proposed concerns about BPA release from dental adhesives ⁵⁴ , ⁵⁵ and other orthodontic materials ⁶ , ⁵⁶ , ⁵⁷ but evidence on safety grounds of clear aligners is inconsistent and not sufficient.

Findings from most of the experimental in vitro studies support the idea that there is no significant estrogenic or cytotoxic capacity of thermoplastic materials, either for as-received or retrieved appliances. Absolutely, the unique environment of the oral cavity such as unpredictable mastication stresses and fluctuating PH and temperature besides the protective and neutralizing effects of saliva which was supported by Premaraj et al.'s study ²² cannot be simulated perfectly in any in vitro experimental study. In the oral cavity, mastication and attrition induced by the consumption of acidic beverages and enzymatic functions might result in clearer aligner abrasion and attrition which lead to more particle release. This is more pronounced in areas such as cusp tips which are exposed to the pressure of antagonist's teeth during mastication. Perhaps, this is why the only clinical trial in this regard showed much greater levels of BPA leach compared to the in vitro studies. ²³

It is supposed that particulate forms of polymers might induce higher biological action relative to bulk materials due to the increased surface-to-volume ratio which tends to increased reactivity with the surrounding environment. ⁶ Therefore, studies such as Ansari et al., ²⁵ Walele et al., ⁴ and Premaraj et al., ²² which considered particulate or powdered forms of aligner materials may be more reflective of the real effect of clear aligners in the oral cavity.

Renewal of aligners in 2-week intervals may expose the patient to another cycle of abrasion and this cumulative cycle may induce the results different from the in vitro studies which consider just a limited range of time. Indeed, studies considering the serial application of new aligners with 2-week intervals in longer periods which can mimic the orthodontic treatment are more reliable and thus recommended. In addition, the oral environment is a physically dynamic media and the gradual force of moving teeth on the clear aligner during orthodontic treatment can be another source of difference between experimental and in vivo studies. Considering all the above-mentioned reasons, the obtained data from these studies should be interpreted with caution.

Currently, a broad spectrum of cytotoxicity and cell viability assays is applied in the fields of toxicology and pharmacology. The choice of the precise assay is crucial in the assessment of obtained results. MTT assay is one of the most commonly used colorimetric assays to evaluate cytotoxicity or cell viability. This assay reveals cell viability through the determination of mitochondrial function by measuring the activity of mitochondrial enzymes such as succinate dehydrogenase. In this assay, MTT is reduced to a purple formazan and light absorbance at a specific wavelength can then quantify this product. This assay is highly reproducible, safe, and more precise than dye exclusion methods; however, experimental studies demonstrated that it tends to produce to false-positive results for viability which induce underestimation of the cytotoxic effect of tested materials. ⁵⁸ , ⁵⁹ MTT formazan is insoluble in water, and it forms purple needle-shaped crystals in the cells. Therefore, before measuring the absorbance, an organic solvent such as dimethyl sulfoxide or isopropanol is required to solubilize the crystals. ⁵⁸ , ⁵⁹ In addition, the cytotoxicity of MTT formazan makes it difficult to remove cell culture media from the plate wells due to floating cells with MTT formazan needles, giving significant well-to-well error. Additional control experiments should be conducted to reduce false-positive or false-negative results that caused by background interference due to the inclusion of particles. This interference could lead to an overestimation of the cell viability. ⁵⁸ , ⁵⁹ Besides, damaged mitochondria may be still able to reduce MTT to formazan crystals. ⁶⁰ , ⁶¹ , ⁶² Loveland revealed that cells with inactivated mitochondria were also able to produce formazan crystals as well as cells with active mitochondria. ⁶³ In the reviewed studies, Martina et al. ²⁶ and Ahamed et al. ²⁴ considered this assay as the only method for the measurement of cell viability and both of the studies revealed no cytotoxicity but this result is prone to all mentioned shortcomings of this assay and should be interpreted with caution. In addition, in their studies, Nemec et al. ²⁷ and Premaraj et al. ²² had considered methods other than MTT and the reduced metabolic function of oral epithelial cells which were exposed to clear aligners and decrease in cell proliferation was demonstrated noting that no cell cytotoxicity was reported. A different result may be expected if these studies were conducted in oral environment conditions.

Another common method for cell viability measurement is the GC-MS analysis. This assay is consisted of a storage condition in the ethanol-water solution which is more aggressive than the oral environment and may not reveal the real behavior of clear aligners. Gracco et al. stated that the environment in this analysis may underestimate the chemical stability of the aligners. ⁷ Despite the aggressive condition of this assay and underestimated chemical stability, no leaching was revealed in Schuster et al. ⁶ and Gracco et al. ⁷ study while the leaching was under the reference dose for daily intake in Kotyk and Wiltshire. ²¹ study.

In some studies, only a single assay was conducted. Considering all mentioned shortcomings of different assays, it is obvious that a single assay is not sufficient to reflect the precise results. Hence, to increase the reliability of obtained results, more than one assay is recommended to be used in in vitro studies for cytotoxicity determination. Materials of aligners like polyurethane affect the expression of many factors involved in the epithelial barrier function and local inflammatory response. ²⁷ The role of these factors in any of the side effects of aligners' therapy has still to be established in clinical studies.

Although according to the EPA, the safe dose of BPA for daily intake is supposed to be 50 μg/kg, ¹⁹ , ⁶⁴ , ⁶⁵ there is evidence which show the human seminoma cell proliferation at low doses of BPA; if the low dose of BPA can cross the placenta, it can interfere with the fetal germ cell proliferation and differentiation. ⁶⁶ In male mice, vom Saal et al. asserted that doses of 0.02, 0.2, and 2.0 Ng/g/day increased adult prostate weight, whereas a 200 Ng/g dose decreased adult prostate weight in male offspring. ⁵² Their study revealed that a small increase in BPA may change prostate cell differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. ⁵² Hence, BPA leaching even in negligible amounts may presume adverse effects and is not definitely safe.

There were considerable variations among the methodologies of the available studies. Still, it seems that quantitatively speaking, the amount of leached BPA reported by in vitro studies is very low, if not zero. However, given the very high levels of BPA leach observed in the only clinical trial and considering other possible dangers of small traces of BPA (even at low doses), as well as noting the frequent adverse events associated with clear aligners and transparent vacuum-formed retainers, it seems that their potential biocompatibility issues should be taken seriously, and therefore, more clinical trials should be conducted to assess the leached BPA amounts and other hazard indicators (such as cytotoxicity) in the oral environment. Such potential effects of BPA even at low doses might describe in part why even despite the observed low amounts of released BPA, many adverse effects were linked to using clear aligners, including pain and soft-tissue issues such as burning, tingling, sore tongue, swelling of lip, blister, ulceration, dry mouth, periodontal problems, and even systemic problems such as difficulty in breathing. Besides these biological adverse effects, oral dysfunctions, and speech difficulties and tooth damages may be associated with clear aligners and should as well be taken into consideration.

Financial support and sponsorship

The study was self-funded by the authors and their institutions.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.