Comparison of translucency, thickness, and gap width of thermoformed and 3D-printed clear aligners using micro-CT and spectrophotometer

The present study compared the thickness and gap width of thermoformed and 3D-printed clear aligners (CAs) using micro-computed tomography (micro-CT) and evaluated their translucency using spectrophotometer. Four groups of CAs were tested: thermoformed with polyethylene terephthalate glycol (TS) or copolyester-elastomer combination (TM), and 3D-printed TC-85 cleaned with alcohol (PA) or with centrifuge (PC). CIELab coordinates were measured (n = 10) to evaluate translucency. CAs (n = 10) were fitted onto respective models and micro-CT was performed to evaluate the thickness and gap width. Thickness and gap width were measured for different tooth type and location in sagittal sections on all sides. The PC group showed significantly higher translucency than the PA group, which was similar to the TS and TM groups (p < 0.01). After the manufacturing process, thickness reduction was observed in the thermoformed groups, whereas thickness increase was observed in the 3D printed-groups. The TM group showed the least gap width amongst the groups (p < 0.01). Thermoformed and 3D-printed CAs had significantly varied thicknesses and regions of best fit depending on the tooth type and location. Differences in the translucency and thickness of the 3D-printed CAs were observed depending on the cleaning methods.

. Median translucency parameter for CAs using different fabrication protocols. p-values were calculated using Kruskal-Wallis test for multiple comparisons, followed by post hoc comparisons using Mann-Whitney U test with a Bonferroni adjustment of alpha level. *p < 0.01.  www.nature.com/scientificreports/ (p < 0.01). The median gap width was significantly higher in the PA, PC groups than in the TM group (p < 0.01) ( Table 2).
Group comparisons for thickness depending on tooth type and location. In the TS, PA, and PC groups, the median thickness was greater for the anterior teeth than for the posterior teeth (TS: p < 0.05; PA and PC: p < 0.01). Group variations in the median thickness were observed as follows: TS: buccogingival < buccal < palatogingival < palatal < incisal or occlusal; TM: buccogingival < buccal < palatogingival < palatal, incisal or occlusal; and PA, PC: buccal, palatogingival, buccogingival < palatal < incisal or occlusal. The thickness variations are further detailed in Table 3 and Fig. 1A,C.
Group comparisons for gap width depending on tooth type and location. In the TM group, the median gap width was greater for the anterior teeth than for the posterior teeth (p < 0.05), while the median gap width for the anterior and posterior teeth were similar in the other groups.

Discussion
Transparency is the physical property that allows light to pass through a material, which is an important factor in determining the esthetics of the CAs and is the major concern of individuals seeking orthodontic treatment 15,16 . The thickness and gap width of CAs are also important factors that can have the greatest influence on orthodontic treatment 10,12,14 . The thickness of CAs can be used as a predictive factor by orthodontist to control the physiological forces and moments applied to the teeth during treatment 33 . During orthodontic treatment, the physiological forces applied to the teeth enable resorptive remodeling and controlled tooth movement 34,35 . However, overloading the teeth with excessive forces can result in side effects, such as cell death, tissue hyalinization of the periodontal ligament, alveolar bone necrosis, and external root resorption 36 . The inner surface of the aligner must be as close to the teeth as possible to provide a clinically effective force 10 . Moreover, a reduced fit Table 2. Median thickness and gap width for CAs using different fabrication protocols. p-values were calculated using Kruskal-Wallis test, followed by post hoc comparisons using Mann-Whitney U test with a Bonferroni adjustment of alpha level. *p < 0.01. There was no significant difference in gap width between the TS and TM group.   www.nature.com/scientificreports/ can cause aligners to lift up during the torquing process of root movements, making it difficult to establish an effective force couple 37 .
In addition, the post-processing procedure is a crucial step as it can affect the printing accuracy and mechanical properties of the printed objects 23,24 . However, there is insufficient evidence-based data to evaluate the changes in the translucency, thickness, and gap width of TC-85 3D-printed CAs according to different cleaning methods.  www.nature.com/scientificreports/ Therefore, this study used IPA and centrifugation as the two chemical and non-chemical cleaning methods for evaluating the outcomes of the 3D-printed samples. Thermoformed CAs made from thermoplastic materials could be worn immediately on the teeth. However, 3D-printed CAs developed using the TC-85 material required a clinically different wearing protocol, as recommended by the manufacturer due to their special properties, which was applied in this study. Based on the geometric stability at high temperatures and shape-memory property of TC-85, 3D-printed CAs were immersed in 80 °C water, which is higher than the glass transition temperature 31 . This immersion was performed to increase flexibility of the aligners before placing them on the models 31 . Afterward, the sample gradually recovered its original shape and fit when kept at a temperature of 37 °C, indicating that the deformation was reversible 31 . In summary, the softening of aligners by warm water provided the comfortable wear to the models. Thereafter, the samples were dried at a temperature of 37 °C to recover their original shape and strength.
There were differences in translucency among the CAs manufactured by different fabrication protocols. The PC group showed significantly higher translucency, similar to that of the TS and TM groups. On the other hand, the PA group showed a decrease in translucency. IPA is the most commonly used solvent for dissolving excess uncured resin 27 . However, previous studies have suggested that IPA, due to low compatibility with acrylate based polymer, tends to cause polymer swelling instead of dissolution 38,39 . Furthermore, as IPA evaporates from the swollen polymer surface, the polymer chains can undergo reorganization, resulting in changes in the surface's solubility and leading to surface cracking of the 3D-printe objects 39 . Therefore, it could be inferred that IPA cleaning has a negative effect on the surface properties of the TC-85 3D-printed CAs.
Further, the findings of our study demonstrated that the thermoformed CAs decreased in thickness after the fabrication process, while the 3D-printed CAs increased in thickness compared to their set-up thickness. While an increase in overall thickness was observed in the PC group compared to the PA group, there was no significant difference in thickness after the thermoforming process of the TS and TM groups. In previous studies, it has been shown that thermoplastic materials experience a decrease in thickness during the thermoforming process, while 3D-printed aligners undergo an increase in thickness 13 Significant differences in thickness for each tooth type were observed among the CAs manufactured by different fabrication protocols. The TS group was less stretched at the anterior teeth than at the posterior teeth under heat and pressure. The PA and PC groups showed thicker output in the anterior teeth compared to the posterior teeth. The reason for the thickening of the anterior teeth in the 3D-printed groups is as follows: the anterior teeth are structurally tapered and have longer crowns. It is difficult to remove any uncured resin remaining on the inner surface before the final post-polymerization step 6 , which may cause an increase in thickness. When observing thickness variations according to tooth location, the TS and TM groups were thinner at the buccal and buccogingival areas. Thermoplastic materials showed a greater thickness reduction in the convex buccal surface and distant gingival areas as they wrapped and extended from the occlusal surface of the model. Further, the median thickness of the incisal/occlusal areas in the PA and PC groups was significantly greater than that in other locations. This appeared to be a printing error owing to the complex curvature of the tooth surface at the incisal edge, occlusal cusp tip, and occlusal central pit. This printing error can occur in the curved contour area during thickness layering, which is affected by the layer thickness, curvature radius, and inclination 40 . www.nature.com/scientificreports/ In the present study, the TM group showed the least gap width amongst the groups. The multi-layer hybrid material consisted of copolyesters and a flexible elastomer core, which has better mechanical strength with a higher maximum load in the tensile test than a single-layer material 5 . Therefore, a multi-layer material might be more extensible during heating and pressure, resulting in a better fit. Different patterns of gap width depending on the tooth location between thermoformed and 3D-printed CAs were also observed. In the thermoformed groups, the incisal/occlusal surface showed the greatest thickness with less variation, but had the largest gap width. In the 3D-printed groups, the buccal side showed the least thickness and variation with a small gap width, whereas the incisal/occlusal surface demonstrated the greatest variation in thickness, resulting in the thickest aligner, but with the smallest gap width. However, the gingival areas on both sides had less thickness variation and showed the largest gap width in the 3D-printed CAs. This phenomenon occurs due to the accumulation of polymerization shrinkage during the printing process, that magnifies the unsuitability of the gingival margins 41 .
Based on previous studies, various parameter optimizations were performed to improve 3D printing accuracy in this study. Firstly, the post-curing process for 3D-printed CAs was performed under nitrogen conditions that prevented the formation of an oxygen inhibition layer, allowing the surface of the specimen to polymerize 42,43 . Injecting inert nitrogen gas to exclude oxygen can also help to achieve good mechanical properties and surface smoothness of the 3D-printed CAs 43 . Secondly, the build angle and layer thickness are particularly important settings for the accuracy of 3D printing. As a result, the 3D-printed CAs were designed at a build angle of 30° and printed using a digital light processing (DLP) 3D printer with a 50 µm resolution, based on previous studies 44, 45 . Thirdly, a 50 µm offset was applied to the inner surface of the 3D-printed CAs to compensate for the thickness change and to enhance the adaptability 46 . A previous study examining the fit of a printed splint showed that splints with 0.05, 0.1, and 0.2 mm offsets can fit the teeth better than those without offset 46 . In addition, the dimensional accuracy of a 3D-printed aligners is affected by factors including light intensity, exposure time, and the properties of printing materials such as light-blocking pigment concentration and light penetration 44,47,48 . Therefore, it is important to calibrate printing parameters to enhance the printing accuracy and minimize variance in each output 49 .
Though the present study presented an objective assessment of differences among four CAs groups, the findings should be interpreted within the limitations. Although it is theoretically possible to set the thickness of 3D-printed CAs for comparison with thermoformed aligners, the recommendations for effective TC-85 3D-printed aligners have a lower limit of 0.5 mm. Therefore, the sample selection criteria were based on clinically recommended preferences. Taking the above into account, we selected a thickness of 0.75 mm (factoring in thickness reduction) for the thermoplastic materials and a thickness of 0.5 mm for our 3D-printed CAs, which is the optimal thickness currently applied in clinical practice. Even though the wearing protocol recommended by the manufacturer was followed to apply the 3D-printed CAs to the models, there were experimental constraints in creating an oral environment at 37 °C temperature with high relative humidity during micro-CT scanning, which may have affected the fit. The present study used only passive-state CAs with a normal occlusion model to obtain objective evaluation data on 3D-printed CAs, as there is no information on how 3D-printed CAs are manufactured and printed depending on tooth type and location. In the future, it will be important to observe differences in thickness and gap width when CAs are activated in various degrees of malocclusion, and to evaluate how attachments affect the thickness and gap width of the aligners. Furthermore, additional evaluation is needed to determine how the thickness and gap width observed in this study affect the clinical performance of achieving the desired tooth movement.

Conclusion
Our null hypothesis that there is no difference in translucency, thickness, and gap width among groups manufactured by different fabrication protocols was rejected in this study. After the manufacturing process, the thermoformed CAs showed a reduction in thickness while the 3D-printed CAs showed an increase in thickness. The PA group showed the lowest translucency, and the TM group had the best fit amongst the groups. The thickness and regions of best fit of the thermoformed and 3D-printed CAs significantly varied depending on the tooth type and location. Additionally, the cleaning methods used in the post-processing of 3D-printed CAs affected their translucency and thickness.

Materials and methods
Sample preparation. A standardized model of the maxillary dental arch in Korean adults with normal occlusion (CON2001-UL-SP-FEM-32, Nissin Dental, Kyoto, Japan) was used and scanned using an intraoral scanner (D250, 3shape, Copenhagen, Denmark) to create an STL file. A standardized model of 60 mm × 50 mm × 20 mm was then printed (S-100, Graphy Inc., Seoul, Korea) using a DLP 3D printer (Asiga MAX™, Asiga, Alexandria, Australia).
Ten CA samples were prepared for each group. Thermoformed CAs were divided into a single-layer group (TS group) with a PETG sheet (Duran, Scheu-Dental, Iserlohn, Germany) and a multi-layer group (TM group) with copolyester in both outer shells and thermoplastic elastomer in the inner shell sheet (CA pro, Scheu-Dental, Iserlohn, Germany). According to the cleaning method, 3D-printed CAs with photo-polymerizable polyurethane resin (TC-85) were divided into two groups: a PA group with isopropyl alcohol cleaning and a PC group with centrifuge cleaning.
A 0.75-mm-thick thermoplastic material was vacuum-thermoformed on a standardized model using a thermomolding caster (Biostar, Scheu-Dental, Iserlohn, Germany) under the thermal deformation conditions recommended by the manufacturer. The thickness of the CAs may be affected by various conditions, such as model preparation, pressure, heat, and positioning of the model on the platform. Therefore, it is important to control these conditions as much as possible during manufacturing 22 www.nature.com/scientificreports/ height of the model from the teeth, and the model was placed at the center of the platform, such that the midpalatal suture was oriented toward the 12 o' clock position. The orientation of the model was kept constant based on the markings for each thermoforming process. As the final step, the CAs were separated from the model and the gingival edge of the aligner was cut and polished. 3D-printed CAs were designed using computer-assisted design software (Deltaface, Coruo, Limoges, France) with a set up thickness of 0.5 mm, offset of 50 μm, and positioned at 30° as a print angulation with minimum strut supports. A DLP 3D printer (SprintRay Pro 95, SprintRay, Los Angeles, CA) was used with a layer thickness of 50 μm. Two different cleaning methods were used to remove any uncured resin from the aligner surfaces. CAs were cleaned either for 1 min with 99.5% IPA or for 6 min and 500 rpm using a centrifuge. The samples were then cured twice for 25 min under nitrogen conditions with ultraviolet light (385-405 nm) using a post-curing chamber (CureM U102H, Graphy Inc., Seoul, Korea). Final cleaning was performed with flowing water and using an ultrasonic cleaner for 3 min at 76-80 °C. The diagram presented in Fig. 3 shows the experimental design.
Thickness and gap width measurement method using micro-CT. The thermoformed CAs were immediately fitted onto the standardized model at room temperature, whereas the 3D-printed CAs were inserted after being smoothly transformed in warm water at 80 °C, following the manufacturer's clinical protocol. Afterwards, the 3D-printed samples were dried at 37 °C to restore their original shape and strength. All aligners (n = 10) were scanned using a high-resolution micro-CT (Skyscan1173, Bruker, MA, USA) at 40 kV, 200 μA, and 34.9 μm of resolution. A total of 40 micro-CTs were obtained, and the target areas of dentition (anterior teeth: the right maxillary central incisor, canine; posterior teeth: the right maxillary first premolar and molar) were re-orientated using Dataviewer software (version 1.5.6.2, Bruker, MA, USA). Slices were obtained using a horizontal plane from the model base and perpendicular to the middle of the line linking the most mesial and distal contact points of the tooth, and were saved by applying the volume of interest (VOI). The images were analyzed using CTAn software (release 2.5, Bruker, MA, USA) at 300× magnification (Fig. 4). Thereafter, the shortest distance of thickness and gap width were measured by projecting a perpendicular line from each reference point tangent. These 5-7 reference points were based on a pilot study and included gingival margins, buccal and palatal midpoints, and incisal/occlusal points (incisal edge, occlusal cusp tips, and central pit) (Fig. 5) 51 . The occlusal surface of the molars includes both the cusp tips and central pit structures, but they are clinically considered as a single plane. Therefore, instead of separating and analyzing their measurement values individually ( Oc 1 , Oc 2 , Oc 3 ), we interpreted them through mean values. A total of 960 points on tooth surfaces were included in the measurements, which were repeated twice at an interval of two weeks by one researcher.
Translucency measurement method using spectrophotometer. Translucency is the difference in the color of a material with a uniform thickness over white and black backgrounds 52 . A spectrophotometer (CM-3500d, Konica Minolta, Tokyo, Japan) was used to calculate the CIELab coordinates of the specimens placed on white and black backgrounds. The window size of the spectrophotometer was 3 mm, and 10 samples with a 3 mm diameter of 0.75-mm-thick thermoformed and 0.5-mm-thick 3D-printed specimens were prepared. The following Eq. (1) was used to determine translucency: Statistical analysis. SPSS software (version 25.0, IBM, NY, USA) was used for statistical analysis of the data. The Shapiro-Wilk test for normality was applied, and the normality of the distribution was rejected. A nonparametric Mann-Whitney U test and Kruskal-Wallis test were performed to compare the median translucency, thickness, and gap width of aligners depending on tooth type and location, followed by Mann-Whitney test with Bonferroni's correction for multiple comparisons. The p-value for statistical significance (conventional level of 0.05) was divided by the number of statistical tests performed.

Data availability
All of the data supporting this work will be made available from the corresponding author upon reasonable request.  www.nature.com/scientificreports/