To investigate the in vitro cytotoxicity of different thermoplastic materials for clear aligners on human primary gingival fibroblasts (HGFs)

Bucci, Rosaria

D'Antò, Vincenzo

Martina, Stefano

Razionale, Armando Viviano

Rongo, Roberto

Valletta, Rosa

Objectives: To investigate the in vitro cytotoxicity of different thermoplastic materials for clear
aligners on human primary gingival fibroblasts (HGFs).
Materials and Methods: Four materials for clear aligners were considered in this study: Duran
(Scheu-Dental GmbH, Iserlohn, Germany), Biolon (Dreve Dentamid GmbH, Unna, Germany),
Zendura (Bay Materials LLC, Fremont, CA, USA), and SmartTrack (Align Technology, San Jose,
CA, USA). Three out of four materials (Duran, Biolon, Zendura) were assessed as thermoformed
and nonthermoformed, whereas the SmartTrack was assessed only as thermoformed. The
samples were placed at 378C in airtight test tubes containing Dulbecco’s Modified Eagle’s Medium
(DMEM; 0.1 mg/mL) for 14 days. The cell viability of HGFs cultured with this medium was assessed
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were
analyzed by means of one-way and two-way analysis of variance and post hoc tests (a 1⁄4 0.05).
Results: Each material exhibited a slight cytotoxic effect after 14 days. The highest cytotoxicity
level on HGFs was achieved by Biolon (64.6% 6 3.3 of cell viability), followed by Zendura (74.4%
6 2.3 of cell viability), SmartTrack (78.8% 6 6.3 of cell viability), and finally Duran (84.6% 6 4 of
cell viability), which was the least cytotoxic. In the comparison between nonthermoformed and
thermoformed materials for Duran, Biolon, and Zendura, the thermoformed materials showed the
highest level of cytotoxicity (P , .001).
Conclusions: Under the experimental conditions of this study, all the materials for clear aligners
presented a slight cytotoxicity. Biolon was the most cytotoxic and the thermoforming process
increased the cytotoxicity of the materials. (Angle Orthod. 0000;00:000–000.

MARTINA, STEFANO

D'Antò, Vincenzo

Archivio della ricerca - Università degli studi di Napoli Federico II

Original ArticleIn vitro cytotoxicity of different thermoplastic materials for clear alignersStefano Martinaa; Roberto Rongoa; Rosaria Buccia; Armando Viviano Razionaleb;Rosa Vallettac; Vincenzo D’Anto`dABSTRACTObjectives: To investigate the in vitro cytotoxicity of different thermoplastic materials for clearaligners on human primary gingival fibroblasts (HGFs).Materials and Methods: Four materials for clear aligners were considered in this study: Duran(Scheu-Dental GmbH, Iserlohn, Germany), Biolon (Dreve Dentamid GmbH, Unna, Germany),Zendura (Bay Materials LLC, Fremont, CA, USA), and SmartTrack (Align Technology, San Jose,CA, USA). Three out of four materials (Duran, Biolon, Zendura) were assessed as thermoformedand nonthermoformed, whereas the SmartTrack was assessed only as thermoformed. Thesamples were placed at 378C in airtight test tubes containing Dulbecco’s Modified Eagle’s Medium(DMEM; 0.1 mg/mL) for 14 days. The cell viability of HGFs cultured with this medium was assessedby the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data wereanalyzed by means of one-way and two-way analysis of variance and post hoc tests (a ¼ 0.05).Results: Each material exhibited a slight cytotoxic effect after 14 days. The highest cytotoxicitylevel on HGFs was achieved by Biolon (64.6% 6 3.3 of cell viability), followed by Zendura (74.4%6 2.3 of cell viability), SmartTrack (78.8% 6 6.3 of cell viability), and finally Duran (84.6% 6 4 ofcell viability), which was the least cytotoxic. In the comparison between nonthermoformed andthermoformed materials for Duran, Biolon, and Zendura, the thermoformed materials showed thehighest level of cytotoxicity (P , .001).Conclusions: Under the experimental conditions of this study, all the materials for clear alignerspresented a slight cytotoxicity. Biolon was the most cytotoxic and the thermoforming processincreased the cytotoxicity of the materials. (Angle Orthod. 0000;00:000–000.)KEY WORDS: Clear aligners; Biocompatibility; Cytotoxicity; OrthodonticsINTRODUCTIONIncreasing demands for esthetic orthodontic treat-ment, not only for adults1 but also for adolescents andchildren,2 have made clear aligner treatments morecommon. Clear aligners are removable trays made ofplastic material that should be worn about 22 hours aday, for 10–14 days each, for the whole duration oftherapy. Thus, they are in close contact with teeth,gingiva, and intraoral fluids for a long time. Clearaligners are used for increasingly complex and,consequently, longer treatments.3,4 The plastic materi-als might be affected by changes due to the oralenvironment and then release molecules that could bedangerous for oral cells; for these reasons, thecytotoxicity of aligner materials should be investigat-ed.5The biocompatibility of orthodontic materials, suchas composite, bonding materials, miniscrews, brack-ets, and archwires,6–9 has been investigated in depth,but only two previous studies focused on the potentialtoxicity of the materials in clear aligners.10,11 Hence, thea Research Fellow, School of Orthodontics, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Naples, Italy.b Associate Professor, Department of Civil and IndustrialEngineering, University of Pisa, Pisa, Italy.c Associate Professor, School of Orthodontics, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Naples, Italy.d Assistant Professor, School of Orthodontics, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Naples, Italy.Corresponding author: Dr Vincenzo D’Anto`, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Via Pansini, 5 - 80131 Naples,Italy(e-mail: vincenzo.danto@unina.it)Accepted: February 2019. Submitted: September 2018.Published Online: April 4, 2019 0000 by The EH Angle Education and Research Foundation,Inc.DOI: 10.2319/091718-674.1 Angle Orthodontist, Vol 00, No 00, 00001cytotoxicity of clear aligners is still a topic of debate dueto the lack of scientific literature and the conflictingresults in the few available studies. Additionally, in thelast decade, several new aligners appeared on themarket and there is a need to assess the cytotoxicity ofthe different materials used by different brands.Hence, the aim of this study was to determine the invitro cytotoxicity of different thermoplastic materials forclear aligners on human primary gingival fibroblasts(HGFs). The null hypothesis was that there is nodifference in the cytotoxicity induced by eluates fromdifferent materials.MATERIALS AND METHODSMaterials, Chemicals, and CellsPrimary HGFs were obtained from oral surgicalprocedures in healthy 20- to 30-year-old patients withtheir informed consent under a protocol approved bythe Ethical Committee of the University of Naples‘‘Federico II’’ (N.226/14). Tissue fragments werewashed twice in phosphate buffered saline (PBS,Carlo Erba Reagents, Milan, Italy) and transferred intotissue culture dishes, placed in a humidified 5% CO2incubator, in Dulbecco’s Modified Eagle Medium(DMEM, Carlo Erba Reagents), supplemented with10% Fetal Bovine Serum (FBS, Carlo Erba Reagents),2 mM glutamine, 100 U/mL of penicillin, and 100 lg/mLof streptomycin at 378C. After 10 days, fragments wereremoved, and released fibroblasts started proliferating.Once confluence was obtained, cells were washedwith PBS and detached from the culture dishes using abrief treatment with trypsin/ethylenediaminetetraaceticacid for 5 minutes and then recultured until a confluentmonolayer was obtained again.Sample PreparationFour materials for clear aligners, Duran (Scheu-Dental GmbH, Iserlohn, Germany), Biolon (DreveDentamid GmbH, Unna, Germany), Zendura (BayMaterials LLC, Fremont, CA, USA), and SmartTrack(Align Technology, San Jose, CA, USA), were consid-ered in this study (Table 1). Three out of the fourmaterials (Duran, Biolon, Zendura) were assessed asthermoformed and not thermoformed, while Smart-Track was tested only as thermoformed.The samples were sterilized following the protocoldefined by the International Standards Organization(ISO) 10993-5 norm (the method recommended by themanufacturer). The samples were immersed in DMEMfor 14 days and stored under stationary conditions at378C in airtight test tubes. The ratio between the weightof the samples and the volume of the dilutions was 0.1g/mL as recommended by the ISO parameters. Foreach clear aligner material, three samples were usedand, after each release interval, the extracts weresterile-filtered to eliminate solid particles and thenstored at -208C until further use.Cell Viability AssessmentMTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyl tetrazolium bromide] (Sigma Chemical Co., Milan,Italy) was used to evaluate cell viability. HGFs wereplanted into 96-well flat-bottomed, tissue cultureplates, with a density of 104 cells/well. After 24 hoursof incubation, the culture medium was replaced with200 lL/well of the extract. After additional 24 hours,the medium was replaced with 100 lL/well of the MTTsolution (1 mg/mL) in PBS, and the cells wereincubated for an additional hour at 378C in a 5%CO2 atmosphere. After the solution was removed,100 lL/well of dimethyl sulfoxide was added and theplates were swirled gently for 10 minutes. The opticaldensity of each well was immediately measured in aspectrophotometer (Sunrise, Team, Mannederf, Zu-rich, Switzerland) at 590 nm. The optical density of thecells cultured in the DMEM medium without any clearaligner material sample extracts was used as anegative control for 100% cell viability and as areference for the determination of the level ofcytotoxicity in the assay; the Para rubber insteadwas used as a positive control.According to Vande Vannet et al.,12 the followingformula was used to calculate the cell viability:Cell viability (%) ¼ (optical density of test group ‚optical density of cellular control group)3 100.Cell viability was then scored according to theclassification of Ahrari et al.:13 More than 90% cell viability: no cytotoxicity 60%–90% cell viability: slight cytotoxicity 30%–59% cell viability: moderate cytotoxicity Less than 30% cell viability: severe cytotoxicityThree independent experiments were performed intriplicate.Table 1. Description of the Tested MaterialsBrand Material Composition SupplierDuran Polyethylene terephthalateglycol (PETG)Scheu-Dental GmbHBiolon Polyethylene terephthalateglycol (PETG)Dreve Dentamid GmbHZendura Polyurethane resin Bay Materials LLCSmartTrack Multilayer aromaticthermoplasticpolyurethane/copolyester.Align TechnologyAngle Orthodontist, Vol 00, No 00, 00002 MARTINA, RONGO, BUCCI, RAZIONALE, VALLETTA, D’ANTO`Statistical AnalysisDescriptive statistics and statistical analysis wereperformed using the Statistical Package for SocialSciences (SPSS 22.0, SPSS IBM, Armonk, NY, USA).Normal distribution of the data was confirmed by theShapiro-Wilk test. Differences between mean valueswere determined by one-way and two-way analysis ofvariance (ANOVA) with Bonferronni post-hoc test. Thelevel of significance was set at P , 0.05.RESULTSThe MTT results and the level of cytotoxicity at eachtime point for thermoformed and non-thermoformedclear aligner materials evaluated are shown in Tables 2and 3. All the materials exhibited a slight cytotoxiceffect after 14 days.Among thermoformed materials, the highest cytotox-icity on HGFs was achieved by Biolon (64.6% 6 3.3 ofcell viability) followed by Zendura (74.4% 6 2.3 of cellviability), SmartTrack (78.8% 6 6.3 of cell viability), andfinally Duran (84.6% 6 4 of cell viability), which was theleast cytotoxic. The positive control showed a cellviability of 12.5% 6 4.3. In the comparison between thenonthermoformed and the thermoformed materials forDuran, Biolon and Zendura, the highest cytotoxicity waspresent in the thermoformed group (P , .001).DISCUSSIONThe present study evaluated the in vitro cytotoxicityof thermoplastic materials used by different brands ofclear aligners after 14 days. To assess the cytotoxicityon cell cultures of the media containing sampleextractions, HGFs were selected because they arethe principal cell line in the oral tissues, together withepithelial keratinocytes, and are clinically exposed tothe potential toxic effects of thermoplastic materialsbecause the aligners contact the gingiva when they areworn. HGFs are among the most used cells to assessbiocompatibility of dental materials and they arerecommended for in vitro evaluation of materials bythe International Standards Organization (ISO).6The exposure time interval was chosen becausepatients usually change aligners after 14 days. Underthe experimental conditions, all the materials showed aslight cytotoxic effect on day 14, with comparable cellviability levels. These results were similar or lower thanthe cytotoxicity level achieved by many other dentalmaterials such as metallic brackets and bands,miniscrews, or bonding materials.6–8On the other hand, there were only two previousstudies in the scientific literature about cytotoxicity ofclear aligner materials. Eliades et al.10 investigated thepossible release of bisphenol-A (BPA) from Invisalignthermoplastic materials. It could produce estrogenicand cytotoxic effects, as demonstrated in other studiesfor adhesives, composite resins, and polycarbonateproducts.14,15 The authors did not find estrogenic andcytotoxic effects on HGFs in contrast with the results ofthe current study in which slight cytotoxicity wasobserved. Nevertheless, it should be considered thatthe SmartTrack material16 was tested, a thermoplasticpolyurethane that, in 2012, replaced the previousmaterial used in the article by Eliades et al.10 Theauthors also reported that the chemical composition ofInvisalign plastic did not have the necessary ingredi-ents to release bisphenol-A.Premaraj et al.11 hypothesized that isocyanate, anoth-er component of Invisalign material, could affect oralhealth. Indeed, contact allergic reactions to isocyanateexposure have been reported.17,18 This study testedcytotoxicity in vitro on epithelial keratinocytes. Inagreement with the current study results, the authorsfound that exposure to Invisalign plastic caused changesin viability, membrane permeability, and adhesion ofTable 2. Descriptive and Inferential Statistics of Cell ViabilityBrandDay 14Mean 6 SD CytotoxicityDuran 84.6 6 4.02*** Aa SlightBiolon 64.6 6 3.31*** B SlightZendura 74.4 6 2.34*** C SlightSmartTrack 78.8 6 6.35*** AC SlightP Value ,.001*** P , .001 indicates statistically significant differencescompared with the negative control.a Different letters in the subgroups indicate the statisticallysignificant differences among materials.Table 3. Descriptive and Inferential Statistics of Cell ViabilityDay 14Thermoformed NonthermoformedP ValueBrand Mean 6 SD Cytotoxicity Mean 6 SD CytotoxicityDuran 83.7 6 5.91*** Aa Slight 94.9 6 5.59 A Slight .002Biolon 67.0 6 3.22*** B Slight 89.1 6 1.77** A Slight ,.001Zendura 73.7 6 3.19*** C Slight 66.9 6 0.16*** C Slight .035** P , .01, *** P , .001 indicate statistically significant differences compared with the negative control.a Different letters in the subgroups indicate the statistically significant differences among materials. P value of the Analysis of Variance.Angle Orthodontist, Vol 00, No 0, 0000CYTOXICITY OF CLEAR ALIGNERS 3epithelial cells in a saline solution environment. Theyalso demonstrated that saliva might offer protection.No previous scientific research compared the cyto-toxicity of different thermoplastic materials of clearaligners. In the current study, the thermoforming effecton the cytotoxicity of plastic materials for clear alignerswas assessed. The thermoformed materials showedmore cytotoxic behavior; however, only three materialswere analyzed in a nonthermoformed state because itwas not possible to obtain the nonthermoformedSmartTrack material. It was previously shown that therelease of monomers by dental composite materialsinduced cytotoxicity,19 so a similar mechanism ofactions by the thermoplastic materials could beassumed. Considering that the polyethylene tere-phthalate released more added substances as thetemperature increased,20 it is possible that the thermo-forming process could increase the release of mono-mers and consequently increase the cytotoxicity.It certainly should be considered that the intraoralenvironment cannot be completely simulated with invitro methodologies.21 It must be emphasized thateluates were obtained under stationary conditions, whileintraoral use of aligners requires that the appliances arewashed every time they are removed from the mouth.Furthermore, intraoral aging affects the properties ofthermoplastic materials of aligners,22 which could alsoinfluence the biocompatibility. However, it is equally truethat the results revealed a slight level of cytotoxicity thatcould be considered clinically irrelevant.CONCLUSIONS In conclusion, under the experimental conditions, allmaterials for clear aligners showed slight cytotoxicity. The thermoforming process increased the cytotoxic-ity of Polyethylene terephthalate glycol materials. Since materials for clear aligners have only shown aslight level of cytotoxicity, their clinical use may beconsidered safe.REFERENCES1. Ziuchkovski JP, Fields HW, Johnston WM, Lindsey DT.Assessment of perceived orthodontic appliance attractive-ness. Am J Orthod Dentofacial Orthop. 2008;133:S68–78.2. Walton DK, Fields HW, Johnston WM, Rosenstiel SF,Firestone AR, Christensen JC. Orthodontic appliancepreferences of children and adolescents. Am J OrthodDentofacial Orthop. 2010;138 698:e1–12.3. Jheon AH, Oberoi S, Solem RC, Kapila S. Moving towardsprecision orthodontics: An evolving paradigm shift in theplanning and delivery of customized orthodontic therapy.Orthod Craniofac Res. 2017;20 Suppl 1:106–113.4. Zheng M, Liu R, Ni Z, Yu Z. Efficiency, effectiveness andtreatment stability of clear aligners: A systematic review andmeta-analysis. Orthod Craniofac Res. 2017;20:127–133.5. Eliades T, Bourauel C. Intraoral aging of orthodonticmaterials: the picture we miss and its clinical relevance.Am J Orthod Dentofacial Orthop. 2005;127(4):403–412.6. D’Anto` V, Spagnuolo G, Polito I, Paduano S, Ambrosio L,Valletta R. In vitro cytotoxicity of orthodontic primers. ProgOrthod. 2009;10:4–11.7. Mockers O, Deroze D, Camps J. Cytotoxicity of orthodonticbands, brackets and archwires in vitro. Dent Mater. 2002;18:311–317.8. Galeotti A, Uomo R, Spagnuolo G, et al. Effect of pH on invitro biocompatibility of orthodontic miniscrew implants. ProgOrthod. 2013;14:15.9. Rongo R, Valletta R, Bucci R, et al. In vitro biocompatibility ofnickel-titanium esthetic orthodontic archwires. Angle Orthod.2016;86:789–795.10. Eliades T, Pratsinis H, Athanasiou AE, Eliades G, Kletsas D.Cytotoxicity and estrogenicity of Invisalign appliances. Am JOrthod Dentofacial Orthop. 2009;136: 100–103.11. Premaraj T, Simet S, Beatty M, Premaraj S. Oral epithelialcell reaction after exposure to Invisalign plastic material. AmJ Orthod Dentofacial Orthop. 2014;145:64–71.12. Vande Vannet B, Mohebbian N, Wehrbein H. Toxicity ofused orthodontic archwires assessed by three-dimensionalcell culture. Eur J Orthod. 2006;28:426–432.13. Ahrari F, Tavakkol Afshari J, Poosti M, Brook A. Cytotoxicityof orthodontic bonding adhesive resins on human oralfibroblasts. Eur J Orthod. 2010;32:688–692.14. Tarumi H, Imazato S, Narimatsu M, Matsuo M, Ebisu S.Estrogenicity of fissure sealants and adhesive resinsdetermined by reporter gene assay. J Dent Res. 2000;79:1838–1843.15. Gioka C, Bourauel C, Hiskia A, Kletsas D, Eliades T, EliadesG. Light-cured or chemically cured orthodontic adhesiveresins? A selection based on the degree of cure, monomerleaching cytotoxicity. Am J Orthod Dentofacial Orthop. 2005;127:413–419.16. Align Technology, Inc. Invisalign introduces Smart-Track.Available at: http://invisalign.com.au/doctor/doc/brochures/SmartTrack_brochure.pdf.17. Frick M, Isaksson M, Bjorkner B, Hindsen M, Ponten A,Bruze M. Occupational allergic contact dermatitis in acompany manufacturing boards coated with isocyanatelacquer. Contact Dermatitis. 2003;48:255–260.18. Hamada S. Dermal uptake study with 4,40-diphenylmethanediisocyanate led to active sensitization. Contact Dermatitis.2012;66:101–105.19. Stanislawsky L, Daniau X, Lauti A, Goldberg M. Factorsresponsible for pulp cell cytotoxicity induced by resin-modified glass ionomer cements. J Biomed Mater Res.1999;48:277–288.20. Bach C, Dauchy X, Severin I, Munoz JF, Etienne S, ChagnonMC. Effect of temperature on the release of intentionally andnon-intentionally added substances from polyethylene tere-phthalate (PET) bottles into water: chemical analysis andpotential toxicity. Food Chem. 2013;139:672–680.21. Rongo R, Ametrano G, Gloria A, et al. Effects of intraoralaging on surface properties of coated nickel-titaniumarchwires. Angle Orthod. 2014;84:665–672.22. Schuster S, Eliades G, Zinelis S, Eliades T, Bradley TG.Structural conformation and leaching from in vitro aged andretrieved Invisalign appliances. Am J Orthod DentofacialOrthop. 2004;126:725–728.Angle Orthodontist, Vol 00, No 00, 00004 MARTINA, RONGO, BUCCI, RAZIONALE, VALLETTA, D’ANTO`

In vitro cytotoxicity of different thermoplastic materials for clear aligners

Objectives: To investigate the in vitro cytotoxicity of different thermoplastic materials for clear aligners on human primary gingival fibroblasts (HGFs).Materials and Methods: Four materials for clear aligners were considered in this study: Duran (Scheu-Dental GmbH, Iserlohn, Germany), Biolon (Dreve Dentamid GmbH, Unna, Germany), Zendura (Bay Materials LLC, Fremont, CA, USA), and SmartTrack (Align Technology, San Jose, CA, USA). Three out of four materials (Duran, Biolon, Zendura) were assessed as thermoformed and nonthermoformed, whereas the SmartTrack was assessed only as thermoformed. The samples were placed at 37 degrees C in airtight test tubes containing Dulbecco's Modified Eagle's Medium (DMEM; 0.1 mg/mL) for 14 days. The cell viability of HGFs cultured with this medium was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by means of one-way and two-way analysis of variance and post hoc tests (alpha = 0.05).Results: Each material exhibited a slight cytotoxic effect after 14 days. The highest cytotoxicity level on HGFs was achieved by Biolon (64.6% +/- 3.3 of cell viability), followed by Zendura (74.4% +/- 2.3 of cell viability), SmartTrack (78.8% +/- 6.3 of cell viability), and finally Duran (84.6% +/- 4 of cell viability), which was the least cytotoxic. In the comparison between nonthermoformed and thermoformed materials for Duran, Biolon, and Zendura, the thermoformed materials showed the highest level of cytotoxicity (P &lt; .001).Conclusions: Under the experimental conditions of this study, all the materials for clear aligners presented a slight cytotoxicity. Biolon was the most cytotoxic and the thermoforming process increased the cytotoxicity of the materials

Archivio della Ricerca - Università di Salerno

OBJECTIVES: To investigate the in vitro cytotoxicity of different thermoplastic materials for clear aligners on human primary gingival fibroblasts (HGFs). MATERIALS AND METHODS: Four materials for clear aligners were considered in this study: Duran (Scheu-Dental GmbH, Iserlohn, Germany), Biolon (Dreve Dentamid GmbH, Unna, Germany), Zendura (Bay Materials LLC, Fremont, CA, USA), and SmartTrack (Align Technology, San Jose, CA, USA). Three out of four materials (Duran, Biolon, Zendura) were assessed as thermoformed and nonthermoformed, whereas the SmartTrack was assessed only as thermoformed. The samples were placed at 37°C in airtight test tubes containing Dulbecco's Modified Eagle's Medium (DMEM; 0.1 mg/mL) for 14 days. The cell viability of HGFs cultured with this medium was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by means of one-way and two-way analysis of variance and post hoc tests (α = 0.05). RESULTS: Each material exhibited a slight cytotoxic effect after 14 days. The highest cytotoxicity level on HGFs was achieved by Biolon (64.6% ± 3.3 of cell viability), followed by Zendura (74.4% ± 2.3 of cell viability), SmartTrack (78.8% ± 6.3 of cell viability), and finally Duran (84.6% ± 4 of cell viability), which was the least cytotoxic. In the comparison between nonthermoformed and thermoformed materials for Duran, Biolon, and Zendura, the thermoformed materials showed the highest level of cytotoxicity (P &lt; .001). CONCLUSIONS: Under the experimental conditions of this study, all the materials for clear aligners presented a slight cytotoxicity. Biolon was the most cytotoxic and the thermoforming process increased the cytotoxicity of the materials

Martina S.

Rongo R.

Bucci R.

Razionale A. V.

Valletta R.

D'Anto V.

Archivio della Ricerca - Università di Pisa

Original ArticleIn vitro cytotoxicity of different thermoplastic materials for clear alignersStefano Martinaa; Roberto Rongoa; Rosaria Buccia; Armando Viviano Razionaleb;Rosa Vallettac; Vincenzo D’AntòdABSTRACTObjectives: To investigate the in vitro cytotoxicity of different thermoplastic materials for clearaligners on human primary gingival fibroblasts (HGFs).Materials and Methods: Four materials for clear aligners were considered in this study: Duran(Scheu-Dental GmbH, Iserlohn, Germany), Biolon (Dreve Dentamid GmbH, Unna, Germany),Zendura (Bay Materials LLC, Fremont, CA, USA), and SmartTrack (Align Technology, San Jose,CA, USA). Three out of four materials (Duran, Biolon, Zendura) were assessed as thermoformedand nonthermoformed, whereas the SmartTrack was assessed only as thermoformed. Thesamples were placed at 378C in airtight test tubes containing Dulbecco’s Modified Eagle’s Medium(DMEM; 0.1 mg/mL) for 14 days. The cell viability of HGFs cultured with this medium was assessedby the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data wereanalyzed by means of one-way and two-way analysis of variance and post hoc tests (a ¼ 0.05).Results: Each material exhibited a slight cytotoxic effect after 14 days. The highest cytotoxicitylevel on HGFs was achieved by Biolon (64.6% 6 3.3 of cell viability), followed by Zendura (74.4%6 2.3 of cell viability), SmartTrack (78.8% 6 6.3 of cell viability), and finally Duran (84.6% 6 4 ofcell viability), which was the least cytotoxic. In the comparison between nonthermoformed andthermoformed materials for Duran, Biolon, and Zendura, the thermoformed materials showed thehighest level of cytotoxicity (P , .001).Conclusions: Under the experimental conditions of this study, all the materials for clear alignerspresented a slight cytotoxicity. Biolon was the most cytotoxic and the thermoforming processincreased the cytotoxicity of the materials. (Angle Orthod. 2019;89:942–945.)KEY WORDS: Clear aligners; Biocompatibility; Cytotoxicity; OrthodonticsINTRODUCTIONIncreasing demands for esthetic orthodontic treat-ment, not only for adults1 but also for adolescents andchildren,2 have made clear aligner treatments morecommon. Clear aligners are removable trays made ofplastic material that should be worn about 22 hours aday, for 10–14 days each, for the whole duration oftherapy. Thus, they are in close contact with teeth,gingiva, and intraoral fluids for a long time. Clearaligners are used for increasingly complex and,consequently, longer treatments.3,4 The plastic materi-als might be affected by changes due to the oralenvironment and then release molecules that could bedangerous for oral cells; for these reasons, thecytotoxicity of aligner materials should be investigat-ed.5The biocompatibility of orthodontic materials, suchas composite, bonding materials, miniscrews, brack-ets, and archwires,6–9 has been investigated in depth,but only two previous studies focused on the potentialtoxicity of the materials in clear aligners.10,11 Hence, thea Research Fellow, School of Orthodontics, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Naples, Italy.b Associate Professor, Department of Civil and IndustrialEngineering, University of Pisa, Pisa, Italy.c Associate Professor, School of Orthodontics, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Naples, Italy.d Assistant Professor, School of Orthodontics, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Naples, Italy.Corresponding author: Dr Vincenzo D’Antò, Department ofNeurosciences, Reproductive Sciences and Oral Sciences,University of Naples Federico II, Via Pansini, 5 - 80131 Naples,Italy(e-mail: vincenzo.danto@unina.it)Accepted: February 2019. Submitted: September 2018.Published Online: April 4, 2019 2019 by The EH Angle Education and Research Foundation,Inc.Angle Orthodontist, Vol 89, No 6, 2019 DOI: 10.2319/091718-674.1942cytotoxicity of clear aligners is still a topic of debate dueto the lack of scientific literature and the conflictingresults in the few available studies. Additionally, in thelast decade, several new aligners appeared on themarket and there is a need to assess the cytotoxicity ofthe different materials used by different brands.Hence, the aim of this study was to determine the invitro cytotoxicity of different thermoplastic materials forclear aligners on human primary gingival fibroblasts(HGFs). The null hypothesis was that there is nodifference in the cytotoxicity induced by eluates fromdifferent materials.MATERIALS AND METHODSMaterials, Chemicals, and CellsPrimary HGFs were obtained from oral surgicalprocedures in healthy 20- to 30-year-old patients withtheir informed consent under a protocol approved bythe Ethical Committee of the University of Naples‘‘Federico II’’ (N.226/14). Tissue fragments werewashed twice in phosphate buffered saline (PBS,Carlo Erba Reagents, Milan, Italy) and transferred intotissue culture dishes, placed in a humidified 5% CO2incubator, in Dulbecco’s Modified Eagle Medium(DMEM, Carlo Erba Reagents), supplemented with10% Fetal Bovine Serum (FBS, Carlo Erba Reagents),2 mM glutamine, 100 U/mL of penicillin, and 100 lg/mLof streptomycin at 378C. After 10 days, fragments wereremoved, and released fibroblasts started proliferating.Once confluence was obtained, cells were washedwith PBS and detached from the culture dishes using abrief treatment with trypsin/ethylenediaminetetraaceticacid for 5 minutes and then recultured until a confluentmonolayer was obtained again.Sample PreparationFour materials for clear aligners, Duran (Scheu-Dental GmbH, Iserlohn, Germany), Biolon (DreveDentamid GmbH, Unna, Germany), Zendura (BayMaterials LLC, Fremont, CA, USA), and SmartTrack(Align Technology, San Jose, CA, USA), were consid-ered in this study (Table 1). Three out of the fourmaterials (Duran, Biolon, Zendura) were assessed asthermoformed and not thermoformed, while Smart-Track was tested only as thermoformed.The samples were sterilized following the protocoldefined by the International Standards Organization(ISO) 10993-5 norm (the method recommended by themanufacturer). The samples were immersed in DMEMfor 14 days and stored under stationary conditions at378C in airtight test tubes. The ratio between the weightof the samples and the volume of the dilutions was 0.1g/mL as recommended by the ISO parameters. Foreach clear aligner material, three samples were usedand, after each release interval, the extracts weresterile-filtered to eliminate solid particles and thenstored at -208C until further use.Cell Viability AssessmentMTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyl tetrazolium bromide] (Sigma Chemical Co., Milan,Italy) was used to evaluate cell viability. HGFs wereplanted into 96-well flat-bottomed, tissue cultureplates, with a density of 104 cells/well. After 24 hoursof incubation, the culture medium was replaced with200 lL/well of the extract. After additional 24 hours,the medium was replaced with 100 lL/well of the MTTsolution (1 mg/mL) in PBS, and the cells wereincubated for an additional hour at 378C in a 5%CO2 atmosphere. After the solution was removed,100 lL/well of dimethyl sulfoxide was added and theplates were swirled gently for 10 minutes. The opticaldensity of each well was immediately measured in aspectrophotometer (Sunrise, Team, Mannederf, Zu-rich, Switzerland) at 590 nm. The optical density of thecells cultured in the DMEM medium without any clearaligner material sample extracts was used as anegative control for 100% cell viability and as areference for the determination of the level ofcytotoxicity in the assay; the Para rubber insteadwas used as a positive control.According to Vande Vannet et al.,12 the followingformula was used to calculate the cell viability:Cell viability (%) ¼ (optical density of test group ‚optical density of cellular control group) 3 100.Cell viability was then scored according to theclassification of Ahrari et al.:13 More than 90% cell viability: no cytotoxicity 60%–90% cell viability: slight cytotoxicity 30%–59% cell viability: moderate cytotoxicity Less than 30% cell viability: severe cytotoxicityThree independent experiments were performed intriplicate.Table 1. Description of the Tested MaterialsBrand Material Composition SupplierDuran Polyethylene terephthalateglycol (PETG)Scheu-Dental GmbHBiolon Polyethylene terephthalateglycol (PETG)Dreve Dentamid GmbHZendura Polyurethane resin Bay Materials LLCSmartTrack Multilayer aromaticthermoplasticpolyurethane/copolyester.Align TechnologyAngle Orthodontist, Vol 89, No 6, 2019CYTOXICITY OF CLEAR ALIGNERS 943Statistical AnalysisDescriptive statistics and statistical analysis wereperformed using the Statistical Package for SocialSciences (SPSS 22.0, SPSS IBM, Armonk, NY, USA).Normal distribution of the data was confirmed by theShapiro-Wilk test. Differences between mean valueswere determined by one-way and two-way analysis ofvariance (ANOVA) with Bonferronni post-hoc test. Thelevel of significance was set at P , 0.05.RESULTSThe MTT results and the level of cytotoxicity at eachtime point for thermoformed and non-thermoformedclear aligner materials evaluated are shown in Tables 2and 3. All the materials exhibited a slight cytotoxiceffect after 14 days.Among thermoformed materials, the highest cytotox-icity on HGFs was achieved by Biolon (64.6% 6 3.3 ofcell viability) followed by Zendura (74.4% 6 2.3 of cellviability), SmartTrack (78.8% 6 6.3 of cell viability), andfinally Duran (84.6% 6 4 of cell viability), which was theleast cytotoxic. The positive control showed a cellviability of 12.5% 6 4.3. In the comparison between thenonthermoformed and the thermoformed materials forDuran, Biolon and Zendura, the highest cytotoxicity waspresent in the thermoformed group (P , .001).DISCUSSIONThe present study evaluated the in vitro cytotoxicityof thermoplastic materials used by different brands ofclear aligners after 14 days. To assess the cytotoxicityon cell cultures of the media containing sampleextractions, HGFs were selected because they arethe principal cell line in the oral tissues, together withepithelial keratinocytes, and are clinically exposed tothe potential toxic effects of thermoplastic materialsbecause the aligners contact the gingiva when they areworn. HGFs are among the most used cells to assessbiocompatibility of dental materials and they arerecommended for in vitro evaluation of materials bythe International Standards Organization (ISO).6The exposure time interval was chosen becausepatients usually change aligners after 14 days. Underthe experimental conditions, all the materials showed aslight cytotoxic effect on day 14, with comparable cellviability levels. These results were similar or lower thanthe cytotoxicity level achieved by many other dentalmaterials such as metallic brackets and bands,miniscrews, or bonding materials.6–8On the other hand, there were only two previousstudies in the scientific literature about cytotoxicity ofclear aligner materials. Eliades et al.10 investigated thepossible release of bisphenol-A (BPA) from Invisalignthermoplastic materials. It could produce estrogenicand cytotoxic effects, as demonstrated in other studiesfor adhesives, composite resins, and polycarbonateproducts.14,15 The authors did not find estrogenic andcytotoxic effects on HGFs in contrast with the results ofthe current study in which slight cytotoxicity wasobserved. Nevertheless, it should be considered thatthe SmartTrack material16 was tested, a thermoplasticpolyurethane that, in 2012, replaced the previousmaterial used in the article by Eliades et al.10 Theauthors also reported that the chemical composition ofInvisalign plastic did not have the necessary ingredi-ents to release bisphenol-A.Premaraj et al.11 hypothesized that isocyanate, anoth-er component of Invisalign material, could affect oralhealth. Indeed, contact allergic reactions to isocyanateexposure have been reported.17,18 This study testedcytotoxicity in vitro on epithelial keratinocytes. Inagreement with the current study results, the authorsfound that exposure to Invisalign plastic caused changesin viability, membrane permeability, and adhesion ofTable 2. Descriptive and Inferential Statistics of Cell ViabilityBrandDay 14Mean 6 SD CytotoxicityDuran 84.6 6 4.02*** Aa SlightBiolon 64.6 6 3.31*** B SlightZendura 74.4 6 2.34*** C SlightSmartTrack 78.8 6 6.35*** AC SlightP Value ,.001*** P , .001 indicates statistically significant differencescompared with the negative control.a Different letters in the subgroups indicate the statisticallysignificant differences among materials.Table 3. Descriptive and Inferential Statistics of Cell ViabilityDay 14Thermoformed NonthermoformedP ValueBrand Mean 6 SD Cytotoxicity Mean 6 SD CytotoxicityDuran 83.7 6 5.91*** Aa Slight 94.9 6 5.59 A Slight .002Biolon 67.0 6 3.22*** B Slight 89.1 6 1.77** A Slight ,.001Zendura 73.7 6 3.19*** C Slight 66.9 6 0.16*** C Slight .035** P , .01, *** P , .001 indicate statistically significant differences compared with the negative control.a Different letters in the subgroups indicate the statistically significant differences among materials. P value of the Analysis of Variance.Angle Orthodontist, Vol 89, No 6, 2019944 MARTINA, RONGO, BUCCI, RAZIONALE, VALLETTA, D’ANTÒepithelial cells in a saline solution environment. Theyalso demonstrated that saliva might offer protection.No previous scientific research compared the cyto-toxicity of different thermoplastic materials of clearaligners. In the current study, the thermoforming effecton the cytotoxicity of plastic materials for clear alignerswas assessed. The thermoformed materials showedmore cytotoxic behavior; however, only three materialswere analyzed in a nonthermoformed state because itwas not possible to obtain the nonthermoformedSmartTrack material. It was previously shown that therelease of monomers by dental composite materialsinduced cytotoxicity,19 so a similar mechanism ofactions by the thermoplastic materials could beassumed. Considering that the polyethylene tere-phthalate released more added substances as thetemperature increased,20 it is possible that the thermo-forming process could increase the release of mono-mers and consequently increase the cytotoxicity.It certainly should be considered that the intraoralenvironment cannot be completely simulated with invitro methodologies.21 It must be emphasized thateluates were obtained under stationary conditions, whileintraoral use of aligners requires that the appliances arewashed every time they are removed from the mouth.Furthermore, intraoral aging affects the properties ofthermoplastic materials of aligners,22 which could alsoinfluence the biocompatibility. However, it is equally truethat the results revealed a slight level of cytotoxicity thatcould be considered clinically irrelevant.CONCLUSIONS In conclusion, under the experimental conditions, allmaterials for clear aligners showed slight cytotoxicity. The thermoforming process increased the cytotoxic-ity of Polyethylene terephthalate glycol materials. Since materials for clear aligners have only shown aslight level of cytotoxicity, their clinical use may beconsidered safe.REFERENCES1. Ziuchkovski JP, Fields HW, Johnston WM, Lindsey DT.Assessment of perceived orthodontic appliance attractive-ness. Am J Orthod Dentofacial Orthop. 2008;133:S68–78.2. Walton DK, Fields HW, Johnston WM, Rosenstiel SF,Firestone AR, Christensen JC. Orthodontic appliancepreferences of children and adolescents. Am J OrthodDentofacial Orthop. 2010;138 698:e1–12.3. Jheon AH, Oberoi S, Solem RC, Kapila S. Moving towardsprecision orthodontics: An evolving paradigm shift in theplanning and delivery of customized orthodontic therapy.Orthod Craniofac Res. 2017;20 Suppl 1:106–113.4. Zheng M, Liu R, Ni Z, Yu Z. Efficiency, effectiveness andtreatment stability of clear aligners: A systematic review andmeta-analysis. Orthod Craniofac Res. 2017;20:127–133.5. Eliades T, Bourauel C. Intraoral aging of orthodonticmaterials: the picture we miss and its clinical relevance.Am J Orthod Dentofacial Orthop. 2005;127(4):403–412.6. D’Antò V, Spagnuolo G, Polito I, Paduano S, Ambrosio L,Valletta R. In vitro cytotoxicity of orthodontic primers. ProgOrthod. 2009;10:4–11.7. Mockers O, Deroze D, Camps J. Cytotoxicity of orthodonticbands, brackets and archwires in vitro. Dent Mater. 2002;18:311–317.8. Galeotti A, Uomo R, Spagnuolo G, et al. Effect of pH on invitro biocompatibility of orthodontic miniscrew implants. ProgOrthod. 2013;14:15.9. Rongo R, Valletta R, Bucci R, et al. In vitro biocompatibility ofnickel-titanium esthetic orthodontic archwires. Angle Orthod.2016;86:789–795.10. Eliades T, Pratsinis H, Athanasiou AE, Eliades G, Kletsas D.Cytotoxicity and estrogenicity of Invisalign appliances. Am JOrthod Dentofacial Orthop. 2009;136: 100–103.11. Premaraj T, Simet S, Beatty M, Premaraj S. Oral epithelialcell reaction after exposure to Invisalign plastic material. AmJ Orthod Dentofacial Orthop. 2014;145:64–71.12. Vande Vannet B, Mohebbian N, Wehrbein H. Toxicity ofused orthodontic archwires assessed by three-dimensionalcell culture. Eur J Orthod. 2006;28:426–432.13. Ahrari F, Tavakkol Afshari J, Poosti M, Brook A. Cytotoxicityof orthodontic bonding adhesive resins on human oralfibroblasts. Eur J Orthod. 2010;32:688–692.14. Tarumi H, Imazato S, Narimatsu M, Matsuo M, Ebisu S.Estrogenicity of fissure sealants and adhesive resinsdetermined by reporter gene assay. J Dent Res. 2000;79:1838–1843.15. Gioka C, Bourauel C, Hiskia A, Kletsas D, Eliades T, EliadesG. Light-cured or chemically cured orthodontic adhesiveresins? A selection based on the degree of cure, monomerleaching cytotoxicity. Am J Orthod Dentofacial Orthop. 2005;127:413–419.16. Align Technology, Inc. Invisalign introduces Smart-Track.Available at: http://invisalign.com.au/doctor/doc/brochures/SmartTrack_brochure.pdf.17. Frick M, Isaksson M, Bjorkner B, Hindsen M, Ponten A,Bruze M. Occupational allergic contact dermatitis in acompany manufacturing boards coated with isocyanatelacquer. Contact Dermatitis. 2003;48:255–260.18. Hamada S. Dermal uptake study with 4,40-diphenylmethanediisocyanate led to active sensitization. Contact Dermatitis.2012;66:101–105.19. Stanislawsky L, Daniau X, Lauti A, Goldberg M. Factorsresponsible for pulp cell cytotoxicity induced by resin-modified glass ionomer cements. J Biomed Mater Res.1999;48:277–288.20. Bach C, Dauchy X, Severin I, Munoz JF, Etienne S, ChagnonMC. Effect of temperature on the release of intentionally andnon-intentionally added substances from polyethylene tere-phthalate (PET) bottles into water: chemical analysis andpotential toxicity. Food Chem. 2013;139:672–680.21. Rongo R, Ametrano G, Gloria A, et al. Effects of intraoralaging on surface properties of coated nickel-titaniumarchwires. Angle Orthod. 2014;84:665–672.22. Schuster S, Eliades G, Zinelis S, Eliades T, Bradley TG.Structural conformation and leaching from in vitro aged andretrieved Invisalign appliances. Am J Orthod DentofacialOrthop. 2004;126:725–728.Angle Orthodontist, Vol 89, No 6, 2019CYTOXICITY OF CLEAR ALIGNERS 945

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In vitro cytotoxicity of different thermoplastic materials for clear aligners

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