Coffee Staining and Simulated Brushing Induced Color Changes and Surface Roughness of 3D-Printed Orthodontic Retainer Material

This in vitro study evaluated the influence of combined coffee staining and simulated brushing-induced color changes and surface roughness on 3D-printed orthodontic retainers. Specimens measuring 10 × 10 × 0.75 mm3 were obtained either by conventional vacuum forming or 3D printing at four print angulations (0°, 15°, 30°, and 45°) (n = 10). The prepared specimens were immersed in a coffee beverage and then mechanically brushed using a simulating device. The specimen’s color difference (ΔE) and surface roughness (Ra) were quantified using a spectrophotometer and a non-contact profilometer, respectively. The highest and lowest mean ΔE values were recorded for the 3D-printed-45° (4.68 ± 2.07) and conventional (2.18 ± 0.87) groups, respectively. The overall mean comparison of ΔE between the conventional and 3D-printed groups was statistically significant (p < 0.01). After simulated brushing, all groups showed a statistically significant increase in the Ra values (p < 0.01). The highest Ra was in the 3D-printed-45° (1.009 ± 0.13 µm) and conventional (0.743 ± 0.12 µm) groups, respectively. The overall ΔE of 3D-printed orthodontic retainers was not comparable to conventional VFRs. Among the different angulations used to print the retainers, 15° angulations were the most efficient in terms of color changes and surface roughness and were comparable to conventional VFRs.


Introduction
The implications of retention after properly performed orthodontic treatment are well understood. The periodontal and gingival tissues, masticatory muscles, occlusal stresses, and tongue, as well as any residual growth of the facial bones, can impact the stable position of the teeth [1,2]. The clinical purpose of retention, the final stage of active orthodontic therapy, is to maintain the teeth that have undergone orthodontic correction in a healthy, functional, and aesthetically pleasing position [1,3,4].
Insufficient data exist to determine which retention method is more appropriate for sustaining the outcome of orthodontic treatment [5]. Although many orthodontists prefer bonded retainers, a combination of fixed and removable retainers is most frequently used in orthodontic retention [6][7][8]. Since fixed retainers have a higher failure rate in the upper jaw than in the lower, most clinicians use maxillary removable and mandibular fixed retainers as their usual retention regimen for optimum relapse prevention [9]. The most popular removable orthodontic retainers, in general, are Hawley and vacuum-formed retainers (VFRs) [10,11]. The clinical performance, cost-effectiveness, aesthetic qualities, ease of fabrication, minimum modification needs, and patient acceptability of VFRs, however, to wear and roughness, which promote biofilm formation, discoloration, loss of surface details, and diminished gloss [16][17][18]. The final effect on material roughness relies on the applied load and brushing duration [34]. The color stability of clear retainers remains a significant consideration for both patients and clinicians [30]. These clear retainers are popular among adults who want a more aesthetic option with less metal exposure [27]. Patient adherence to retainer wear may be hampered by material degradation, surface roughness, and discoloration [27].
The precision and properties of 3D-printed resin materials are influenced by many factors, including resin type, printer used, surface treatment, and printing angulations [35]. Although the printing angulation of 3D-printed resin materials has found considerable importance among dental researchers, it is still unclear about the specific printing angulation to be applied for 3D printing [14,16,[36][37][38][39][40][41][42]. Three-dimensional-printed orthodontic retainers are still a relatively new topic in the orthodontic specialty, necessitating a thorough analysis of the properties before considering it a viable option to conventional copolyester VFRs. To date, the effect of print angulation on the ∆E and Ra of 3D-printed orthodontic retainer materials remains unexplored. Therefore, this in vitro study aimed to evaluate the influence of printing angulation, coffee staining, and simulated brushing-induced color changes and surface roughness of 3D-printed orthodontic retainer and compare the outcome with the conventional thermoplastic orthodontic retainer. The first null hypothesis stated was that there is no significant difference in color and surface roughness between the 3D-printed and conventional orthodontic retainers. The second null hypothesis stated was that the four different printing angulations (0 • , 15 • , 30 • , and 45 • ) would present no significant differences in the color and surface roughness when printing 3D-printed orthodontic retainers.
The specimens were printed at four print angulations: 0 • , 15 • , 30 • , and 45 • (n = 10) ( Figure 1B) using a 100 mm layer-by-layer print thickness. Subsequently, the printed specimens were rinsed in two consecutive baths of 98% isopropyl alcohol for 5 min and rigorously dried with compressed air. Then the specimens were post-print cured for 30 min in a curing oven to ensure that the remaining monomers were cured. Print supports from printed specimens were removed carefully before color and roughness measurements.
The conventional retainer specimens were prepared by heating and vacuum forming of the copolyester thermoplastic sheets (Proform, Keystone Industries, Myerstown, PA, USA) over a steel block of a predetermined dimension using a thermoforming machine (Henry Schein Inc., Melville, NY, USA). The conventional retainer specimens were prepared by heating and vacuum fo of the copolyester thermoplastic sheets (Proform, Keystone Industries, Myerstow USA) over a steel block of a predetermined dimension using a thermoforming ma (Henry Schein Inc. Melville, NY, USA).

Coffee Staining and Simulated Brushing
Three teaspoons of coffee powder (Nescafe Classic, Nestle Middle East Manuf ing L.L.C.-Dubai, United Arab Emirates) were added to 250 mL of boiling water an tinuously swirled for 10 min to prepare the coffee beverage. The resulting solutio filtered to remove the residue, then cooled to room temperature. The specimens thoroughly cleaned under running water to remove debris before coffee staining specimens were continually immersed in Petri dishes containing ample amounts coffee beverage. The beverage was changed every 4 h over the 50 h total immersi riod, which equated to two years of oral exposure [17,43].
Before simulating mechanical brushing, the specimens were rinsed with tap and stored in distilled water at room temperature for 24 h. The simulating devic Mechatronik GMBH, Feldkirchen Westerham, Germany) had 12 separate slots, each ing a soft toothbrush (Colgate 360, Colgate-Palmolive, Istanbul, Turkey). A drop of sive monomer was used to glue the specimens to the dentifrice containers of the brush simulator. A 1:1 mixture of dentifrice (Colgate Regular, Colgate-Palmolive A L.T.D, Dammam, Kingdom of Saudi Arabia) and deionized water was prepare poured into each of the 12 containers to immerse the specimens. The brushing pa ters-356 rpm, 2N vertical load, 38 mm stroke length, and 35,600 brushing cyclesapplied. The brushing cycles were accomplished in 100 min, representing two ye oral exposure [17,44]. The brushing force was delivered in accordance with ISO 1 specification, which specifies 0.5-2.5 N brushing forces [45]. According to the AD ommendations, the toothbrushes were replaced after 4500 cycles, and the slurry w plenished during the brushing cycle [17].

Color
A bench-top UV light visible spectrophotometer (Konica Minolta Sensing Osaka, Japan) functioning at a wavelength between 360 and 740 nm was used to r the color of the specimens before and after coffee staining and brushing in the 3D mission Internationale de l'Eclairege (CIE) Lab color space. Following the manufact recommendation, the spectrophotometer was calibrated against a white backgroun

Coffee Staining and Simulated Brushing
Three teaspoons of coffee powder (Nescafe Classic, Nestle Middle East Manufacturing L.L.C.-Dubai, United Arab Emirates) were added to 250 mL of boiling water and continuously swirled for 10 min to prepare the coffee beverage. The resulting solution was filtered to remove the residue, then cooled to room temperature. The specimens were thoroughly cleaned under running water to remove debris before coffee staining. The specimens were continually immersed in Petri dishes containing ample amounts of the coffee beverage. The beverage was changed every 4 h over the 50 h total immersion period, which equated to two years of oral exposure [17,43].
Before simulating mechanical brushing, the specimens were rinsed with tap water and stored in distilled water at room temperature for 24 h. The simulating device (SD Mechatronik GMBH, Feldkirchen Westerham, Germany) had 12 separate slots, each holding a soft toothbrush (Colgate 360, Colgate-Palmolive, Istanbul, Turkey). A drop of adhesive monomer was used to glue the specimens to the dentifrice containers of the toothbrush simulator. A 1:1 mixture of dentifrice (Colgate Regular, Colgate-Palmolive Arabia L.T.D, Dammam, Kingdom of Saudi Arabia) and deionized water was prepared and poured into each of the 12 containers to immerse the specimens. The brushing parameters-356 rpm, 2N vertical load, 38 mm stroke length, and 35,600 brushing cycles-were applied. The brushing cycles were accomplished in 100 min, representing two years of oral exposure [17,44]. The brushing force was delivered in accordance with ISO 14569-1 specification, which specifies 0.5-2.5 N brushing forces [45]. According to the ADA recommendations, the toothbrushes were replaced after 4500 cycles, and the slurry was replenished during the brushing cycle [17].

Color
A bench-top UV light visible spectrophotometer (Konica Minolta Sensing Inc., Osaka, Japan) functioning at a wavelength between 360 and 740 nm was used to record the color of the specimens before and after coffee staining and brushing in the 3D Commission Internationale de l'Eclairege (CIE) Lab color space. Following the manufacturer's recommendation, the spectrophotometer was calibrated against a white background. CIE color coordinates were measured using three-pulsed xenon lamps analogous to average daylight.
According to the International Organization for Standardization (ISO) technical specification ISO/TR 28642:2016 [46], the color difference (∆E) of the specimen was quantified using the CIELab equation: ∆L*, ∆a*, and ∆b* are the mean differences between the pre and post-coffee staining and brushing L*, a*, and b* values. L refers to the difference between light and dark, a is the difference in the red and green chromatic scale, and b is the difference in the quantity of yellow and blue.
In dentistry, the stated values for the CIELab 50% perceptibility threshold (PT) and acceptability threshold (AT) are ∆E = 1.2 and ∆E = 2.7, respectively. The ∆E values over the AT limit are clinically unacceptable [47].
In relating the ∆E to the clinical situation, the obtained values were converted to the National Bureau of Standards (NBS) units using Equation (2). The inferences of color changes as per NBS units are presented in Table 1 [48]:

Surface Roughness
The surface roughness of the specimens was measured using a non-contact optical profilometer (Bruker Contour GT, Tucson, AZ, USA). The profilometer includes a fully automated turret and a nano-lens atomic force microscopy (AFM) module. The specimen was mounted on the turret and scanned using white light interferometry without contacting the surface to measure the roughness. The profilometer equipped with Vision 64 software manages turret movement [35,49]. The specimen was scanned at three different regions, 2 mm apart, and the mean of the readings from the three scans corresponds to the roughness values of that particular specimen. The roughness average (Ra) in µm is used to quantify the roughness. Surface roughness measurement was performed at two intervals, before (Ra1) and after tooth brushing (Ra2).

Scanning Electron Microscopy (SEM) Analysis
A representative specimen from each group was observed under a scanning electron microscope (JEOL JSM-5900 LV SEM, Tokyo, Japan). Before the SEM analysis, the specimen was gold sputter coated for one minute in a coating machine (Q150R, Quorum Technologies, East Sussex, UK). The SEM micrographs were obtained at a magnification of ×1000, 10 kV in a vacuum, and a working distance of 10 µm. Figure 2 presents the flowchart illustrating the study process.

Statistical Analysis
Data were analyzed using SPSS analysis software (SPSS IBM v.22; IBM Corp, Armonk, NY, USA). Data followed a normal distribution (Shapiro-Wilk test; (p > 0.05)). Analysis of Variance (ANOVA) was used to find the significant difference between the groups. Post hoc Bonferroni multiple comparisons test was applied to find significant differences between the groups. Paired t-tests were used to compare the pre and post-brushing surface roughness for each group (α = 0.05).

Statistical Analysis
Data were analyzed using SPSS analysis software (SPSS IBM v.22; IBM Corp, Armonk, NY, USA). Data followed a normal distribution (Shapiro-Wilk test; (P > 0.05)). Analysis of Variance (ANOVA) was used to find the significant difference between the groups. Post hoc Bonferroni multiple comparisons test was applied to find significant differences between the groups. Paired t-tests were used to compare the pre and post-brushing surface roughness for each group (α = 0.05). Figure 3 presents the mean ΔE of the group according to the PT and AT limit. The higher and lower mean color changes were recorded in the 3D-printed-45° group (4.68 ± 2.07) and conventional VFRs group (2.18 ± 0.87), respectively. Among the tested materials, the ΔE values were above the AT limit except for the conventional VFRs, which exhibited ΔE values between the PT and AT limits.  Figure 3 presents the mean ∆E of the group according to the PT and AT limit. The higher and lower mean color changes were recorded in the 3D-printed-45 • group (4.68 ± 2.07) and conventional VFRs group (2.18 ± 0.87), respectively. Among the tested materials, the ∆E values were above the AT limit except for the conventional VFRs, which exhibited ∆E values between the PT and AT limits.

Color Change (∆E)
Pairwise comparison for significant differences in ∆E among the study groups was carried out using Bonferroni multiple comparisons test (Table 2). A statistically significant difference was observed between conventional VFRs and 3D-printed-45 • groups (p < 0.01). No significant difference was observed among the other groups (p > 0.05).
The overall mean comparison of ∆E between the conventional and 3D-printed groups showed a significant difference (p < 0.01) ( Table 3). Figure 4 presents the NBS inference of the ∆E values. Conventional VFRs and 3Dprinted-15 • groups showed appreciable/marked color changes, while the remaining study groups showed noticeable/perceivable color changes.  Pairwise comparison for significant differences in ΔE among the study groups was carried out using Bonferroni multiple comparisons test (Table 2). A statistically significant difference was observed between conventional VFRs and 3D-printed-45° groups (P<0.01). No significant difference was observed among the other groups (P>0.05). The overall mean comparison of ΔE between the conventional and 3D-printed groups showed a significant difference (P< 0.01) ( Table 3).     Figure 5 presents the mean Ra1 and Ra2 of the groups according to the roughness threshold limit. Mean Ra1 was found to be high in 3D-printed-45 • (0.288 ± 0.05) and low in 3D-printed-15 • (0.229 ± 0.08) groups. The difference in mean Ra1 amongst the groups was not statistically significant (p > 0.05). Figure 4 presents the NBS inference of the ΔE values. Conventional VFRs and 3Dprinted-15° groups showed appreciable/marked color changes, while the remaining study groups showed noticeable/perceivable color changes.  Figure 5 presents the mean Ra1 and Ra2 of the groups according to the roughness threshold limit. Mean Ra1 was found to be high in 3D-printed-45° (0.288 ± 0.05) and low in 3D-printed-15° (0.229 ± 0.08) groups. The difference in mean Ra1 amongst the groups was not statistically significant (P > 0.05).
Since the Ra1 between groups was statistically non-significant (p = 0.37), the pairwise comparison was not performed. Pairwise comparison for a significant difference in Ra2 among the groups was carried out using Bonferroni multiple comparisons test (Table 4). A statistically significant difference was observed between conventional VFRs and the 3Dprinted-0 • group (p < 0.01), conventional VFRs and the 3D-printed-30 • group (p < 0.001), conventional VFRs and the 3D-printed-45 • group (p < 0.001), 3D-printed-0 • , and 3Dprinted-15 • group (p < 0.01), 3D-printed-15 • and 3D-printed-30 • group (p < 0.01) as well as between 3D-printed-15 • , and 3D-printed-45 • group (p < 0.001). No significant difference was observed among the other groups (p > 0.05).  Table 5 presents the mean comparison in roughness from Ra1 to Ra2. There was an increase in surface roughness from Ra1 to Ra2 in all the groups, and this increased roughness was statistically significant (p < 0.001). The overall mean comparison of Ra2 between the conventional VFRs and 3D-printed groups was statistically significant (p < 0.01) ( Table 6). Profilometer images of the representative specimen from each study group are presented in Figure 6. At Ra1, the specimens from the study groups ( Figure 6A-E) showed an identical roughness profile, which is consistent with the Ra1 values of the respective group. On the contrary, at Ra2, all the specimens ( Figure 6A1-E1) demonstrated variations in the form of deep scores and uneven surfaces, demonstrating increased roughness due to brushing.  SEM photomicrographs of the representative conventional and 3D-printed specimen groups are presented in Figure 7. The SEM micrographs showed significant changes from Ra1 ( Figure 7A-E) to Ra2 ( Figure 7A1-E1). The surface topographic changes were consistent with that of the profilometric images of the groups. Surface changes at Ra2 in a series of grooves were evident on all the sample surfaces. The 3D-printed (0 • , 30 • , and 45 • ) specimens showed more prominent topographic changes compared to the other two groups.

Discussion
This in vitro study aimed to evaluate the influence of printing angulation, coffee staining, simulated brushing-induced color changes, and surface roughness of 3D-printed orthodontic retainers to compare the outcome with the conventional thermoplastic orthodontic retainer. It was hypothesized that there would be no significant difference in ∆E and Ra between the 3D-printed retainers and conventional VFRs. The study's results demonstrated a significant difference (p < 0.01) in the ∆E between conventional VFRs and the 3D-printed-45 • group (p < 0.01) only, and the mean Ra of the conventional VFRs was significant (p < 0.01) with the 3D-printed groups except for 3D-printed-15 • group (p > 0.01). This outcome suggests partial rejection of the first null hypothesis.
Long-term retainer use is the only method that prevents orthodontic relapse and produces a stable outcome following orthodontic treatment [2,9]. Due to the aesthetic aspects and reasonable treatment times, clear retainers have become popular as orthodontic procedures have advanced. These clear retainers must be maintained to prevent loss of material integrity because interactions between clear retainer material and the oral environment can result in plaque and calculus formation and bacteria buildup and retention on the retainer surface [50]. In the current study, copolyester thermoplastic sheets were used to fabricate the conventional VFRs and compare them with 3D-printed retainers. Copolyester is a result of the modifications of polyester, such as polyethylene terephthalate, with isophthalic acid or other diols. They are shown to wear less and are more transparent than polypropylene polymers [51].
Color stability is an important property for a dental appliance, especially for clear or esthetic appliances. The color is affected by intra-oral aging, ultraviolet radiation, mouthwash, and various dietary beverages [50]. In this study, the retainers were immersed in coffee beverages, partly due to its chromogenic nature of inducing discoloration and because it is a commonly consumed beverage worldwide [17]. The perceptibility threshold (PT) and the acceptability threshold (AT) are crucial for evaluating ∆E. The ∆E value of ≤1 is visually undetectable in a typically controlled environment. Per the ISO/TR-28642:2016 norms, the PT (∆E ≤ 1.2) and AT (∆E = 1.2-2.7) were used to differentiate the ∆E values in this study. Clinically unacceptable ∆E values were those that exceeded the AT limit (∆E ≥ 2.7) [17].
The comparison of the mean ∆E values showed that ∆E of conventional VFR was between the PT and AT (∆E = 2.18), and the ∆E of 3D-printed groups (irrespective of the printing angulation) was above AT (∆E > 2.7). The obtained ∆E values were converted to NBS units in relating the ∆E values to clinical conditions. Among the groups, conventional VFR and 3D-printed-15 • demonstrated appreciable color changes (1.5-3.0 NBS units), while the remaining 3D-printed groups showed noticeable color changes (3.0-6.0 NBS units). The difference in ∆E between the conventional and 3D-printed groups may be due to the 3D-printed resins' surface degradation, which may have affected ∆E. Surface degradation and filler content are inversely correlated, and most 3D-printed resins have less inorganic fillers [17]. On the contrary, excellent transparency, appropriate flow properties, durability, and high chemical change resistance of the copolyester retainer materials could have contributed to higher color stability than 3D-printed groups [52].
The outcome of the study also demonstrated that an increase in surface roughness (Ra2) of the specimens presented with increased color changes. This correlation could be possibly explained by the surface properties of the materials, such as roughness, which accelerate pigment deposition and staining on the specimen surface, thus implicating the differences in the color changes [53]. In order to avoid the accumulation of plaque and bacteria, dental materials should have mean Ra values below 0.2 µm [54]. Plaque accumulates more quickly when surface roughness increases over time. According to studies, if the roughness value surpasses 0.5 µm, an intraoral hard surface can be uncomfortable and felt by the human tongue [55]. The profilometer is the most popular method among dental researchers for measuring surface roughness since it provides a quantitative assessment of surface topography. The common roughness parameter in general quality control is the average arithmetic height (Ra), which is easy to define and compute and gives a reasonable idea of height variations. However, still, comprehensive specimen surface details were provided in this study by combining quantitative measures with qualitative SEM examination [35].
While the current tested materials underwent the same finishing and polishing procedure, there was no discernible difference between them at Ra1. However, roughness (Ra2) significantly increased above the clinical perceptibility threshold (Ra = 0.5 µm) following immersion in coffee beverages and simulating brushing. Toothbrush abrasion is used to evaluate the behavior of dental materials by analyzing the surface changes induced by brushing in an experimental setting [56]. The most popular, easy, and cost-effective mechanical approach to plaque control by an individual is tooth brushing with dentifrice. Contrarily, using dentifrice to brush has been associated with roughening the surfaces of dental hard tissues and restorative and prosthetic dental materials [56]. According to the Relative Dentin Abrasivity (RDA), the Colgate Regular dentifrice used in this research has a mild abrasive (RDA = 68) action. Consequently, it is unrealistic to imply that the low abrasive toothpaste impacted the obtained Ra2 values [35].
The roughness changes could be related to hydrolytic degradation when polymer materials absorb water or other liquid from the atmosphere. Water or beverages and the polymer matrix react chemically during hydrolysis, changing the structure and characteristics of the polymer matrix in many ways. The polymers are irreversibly damaged, and all these modifications impact material roughness [48]. Furthermore, other factors such as material structure, composition and properties, liquid polarity, exposure time of liquids, and pH could all influence the surface roughness of the materials. The increase in surface roughness from Ra1 to Ra2, as confirmed by the profilometer, is well corroborated with the SEM micrographs. The outcome of the study also demonstrated that an increase in surface roughness (Ra2) of the specimens correlated with increased color changes.
The second null hypothesis of this study was that the four different printing angulations (0 • , 15 • , 30 • , and 45 • ) would present no significant differences in the ∆E and Ra when printing orthodontic retainers. The study's outcome demonstrated no significant difference in ∆E among the four printing angulations. Nonetheless, the Ra2 of the 3D-printed-15 • group was significant with other 3D-printed groups (p < 0.01), which suggests partial rejection of the second null hypothesis.
For obtaining precision, smoothness, and accuracy when printing dental models, a print angulation of <30 is suggested by the manufacturer [14,37]. Nonetheless, the configuration must be positioned as nearly vertically as feasible in order to fabricate the maximum number of models efficiently during one print cycle [14]. In general, the shape and printing direction affect the number of models that can be planted on a platform; vertical printing can create more models than horizontal printing [16]. Previous studies have shown mixed outcomes with regard to the accuracy of print angulations.
Williams et al. [14] investigated the precision and accuracy of 3D-printed retainers at different angulations (15 • , 30 • , 45 • , 60 • , and 90 • ) and the impact of angulation on printing time and resin consumption. When compared to the digital reference file, it was found that 3D-printed retainers were accurate to within 0.25 mm at all print angulations at the cusp tips and incisal edges. Smooth facial surfaces extended beyond clinically acceptable levels. The most time and money-efficient printing angulations were 15 and 45 degrees, respectively. McCarty et al. [40] evaluated the impact of print orientation (horizontal, vertical, and 45 • ) on the dimensional accuracy of 3D-printed clear aligners. Under the testing conditions, the authors concluded that print orientation had no significant effect on the overall accuracy of the 3D-printed aligner design. Boyer et al. [41] investigated the effect of print orientation (0 • , 45 • , 90 • , 135 • , 180 • , 225 • , 270 • , and 315 • ) on the dimensional accuracy of 3D-printed orthodontic aligners. Printing at 90 • angulation provided the most accurate prints compared to the other seven orientations tested, albeit not all differences were statistically significant.
In this study, 15 • print angulation was efficient in providing specimens with high color stability and low roughness compared to other angulations. A 45 • print angulation provided low color stability and high roughness values. Shim et al. [36] evaluated the roughness of 3D-printed PMMA specimens in three printing orientations (0 • , 45 • , and 90 • ). The authors found significantly low roughness values for specimens printed at 0 • and 90 • compared to specimens printed at 45 • .
To the authors' knowledge, this is the first study to evaluate the color and roughness of 3D-printed retainer materials in four different printing angulations. Although the study outcome fills an important aspect in the literature related to the color changes and roughness of 3D-printed retainer materials, this study has a few limitations. Foremost, the in vitro protocol of the study is the limitation, despite the intra-oral simulation being as close as possible. The surface treatment in this study followed a one-after-the-other approach (e.g., immersion followed by brushing), whereas this process is simultaneous in the oral cavity. The influence of intra-oral conditions could present a more deviated outcome considering the actions of saliva, personalized oral hygiene habits, and diet. The exact composition of the 3D-printed resins is yet to be available due to the manufacturers' trade secrets, which hinders drawing conclusions about the observed differences. The specimens were prepared flat and did not follow the retainer shape, another limitation of this study. Data regarding the color and roughness of 3D-printed orthodontic retainer materials are scarce, making the comparison of the present result with previous data challenging.
A previous study has shown that the long-term flexibility of the retainer materials is influenced by toothbrushing [51]. Hence it is imperative to study the flexural strength and modulus of the 3D-printed retainer materials at different angulations after toothbrushing. The 3D-printed retainer materials' anti-microbial activity related to printing directions should be tested. Furthermore, the effect of different beverages on the 3D-printed retainer materials could provide a broader understanding of the color and roughness results. It is also worth studying the action of different chemical cleansers or destaining agents on the color and roughness of the 3D-printed retainer materials.

Conclusions
Considering the study's limitations, the following conclusions are made: The overall color stability of 3D-printed orthodontic retainers significantly differed from that of conventional VFRs. The conventional and 3D-printed-15 • groups demonstrated appreciable color changes, while the other 3D-printed groups showed noticeable color changes per the NBS units.
The surface roughness of 3D-printed orthodontic retainers was not comparable to that of conventional VFRs, except for the 3D-printed-15 • group after coffee immersion and simulated brushing. The conventional and 3D-printed groups exceeded the clinical perceptible roughness threshold (Ra > 0.5 µm) after coffee immersion and simulated brushing.
Among the different angulations used to print the 3D-printed retainers, the 15 • angle was the most efficient in terms of color stability and surface roughness and was comparable to conventional VFRs.