Post-processing effects of Aloe vera on biofilm formation and physical properties of 3D-printed dental resins

Abstract

The study aims to investigate the post-processing effects of Aloe vera gel (AV) on biofilm formation and the physical properties of 3D-printed dental resins. Samples manufactured using Biomed Clear Resin were assigned to an Aloe vera-treated group (Group AV) and a control group (Group C), with a total of 128 disk-shaped specimens (n = 64 per group). Group AV underwent post-polymerization in 100% Aloe vera gel, while Group C was directly polymerized. For color stability evaluation, specimens were immersed in staining solutions and incubated at 37 °C for 30 days. Surface morphology (SEM/EDX), water sorption (WSP), and water solubility (WSL) were evaluated. Biofilm formation was assessed using Streptococcus mutans. Intergroup differences were analyzed using the Kruskal–Wallis test, followed by Mann–Whitney U tests for post hoc comparisons (p < .05). Group AV was demonstrated to mitigate color alteration, particularly in coffee and red wine solutions, and SEM analysis revealed a microtextured surface structure. Aloe vera post-processing reduced water solubility, while no significant effects were observed on water sorption or biofilm formation. The results indicate that AV treatment improved the physicochemical stability of 3D-printed dental resins, although no significant antibacterial effect was observed. Aloe vera post-processing may represent a promising natural approach to enhance the longevity and aesthetic performance of 3D-printed dental appliances; however, further clinical validation is required.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-39650-w.

Introduction

In dentistry, additive manufacturing commonly known as 3D printing is extensively used for creating diagnostic models, stabilization splints, clear aligners, surgical guides, and removable appliances¹. Among the different 3D printing techniques, vat photopolymerization, materials cured with a light source may develop an unpolymerized oxidation layer forming due to air exposure during polymerization, limiting conversion to approximately 50%². This layer weakens physical properties and promotes residual monomer release. Post-polymerization methods such as ultrasonic cleaning with alcohol-based solutions or UV light exposure are employed to enhance polymerization and eliminate the oxygen inhibition layer (OIL). During free-radical polymerization, oxygen scavenges active radicals at the surface, creating an OIL that limits conversion and leaves a soft, under-cured interface. Post-curing under an oxygen-barrier medium is a recognized approach to suppress OIL and increase the degree of conversion. Aloe vera gel (AV), which behaves as a low-permeability coating in other contexts, was evaluated here as a natural barrier medium to mitigate OIL during post-curing^3,4. A glycerin-containing gel is commonly applied to improve conversion, leading to higher flexural strength, reduced roughness, increased wear resistance, and diminished discoloration^5,6. More recently, AV has emerged as a promising alternative, not only removing the oxidation layer but also offering anti-inflammatory, antimicrobial, and regenerative benefits^7–9.

AV is utilized in various medical treatments due to its healing, nourishing, antifungal, and antibacterial effects. Its protective and antimicrobial properties allow it to be used as a surface coating for food products, effectively extending shelf life by reducing oxygen permeability¹⁰. In dentistry, AV is used as toothpaste, mouthwash, and gel, with studies demonstrating its effectiveness in healing oral wounds and its anti-cariogenic effects^7,8. Babae et al., have reported that the use of 2% AV resulted in accelerated healing of minor recurrent aphthous stomatitis lesions and reduced pain levels⁸. Similarly, Jain et al. found that high concentrations of AV effectively eliminated oral pathogens responsible for subgingival calculus, periapical, and periodontal abscesses⁷. Despite the extensive use of AV in medical and dental applications, its role as a post-processing agent for enhancing the various properties of 3D-printed materials remains underexplored. In this context, AV was chosen to eliminate the OIL and to explore its effects as a post-processing agent on the physicochemical properties (color stability, water sorption/solubility) and biofilm formation of 3D-printed resins. In the present study, AV was evaluated as a natural oxygen-barrier post-curing medium hypothesized to reduce the OIL.

Processing and post-processing methods influence surface characteristics, color stability, water sorption, and antimicrobial properties⁴. Aesthetic concerns are also critical, as patients of all ages seek visually appealing dental applications. Color stability is a key factor, as discoloration in orthodontic appliances can indicate weak mechanical properties, such as increased water sorption. Additionally, appliances with poor color stability and inadequate mechanical strength are unsuitable for long-term use¹¹. Dental plaque is a complex biofilm containing Streptococcus mutans (S. mutans), the primary cause of caries due to its production of short-chain carboxylic acids that dissolve dental tissues¹². Bacteria that cause caries become more resistant to immune system cells and antimicrobial agents due to biofilm formation^13,14. Bacterial adhesion, the first step of biofilm formation, is facilitated by orthodontic appliances, which provide new surfaces for colonization¹². Material choice influences bacterial adhesion, plaque accumulation, and caries risk¹⁵. Post-polymerization may also impact antimicrobial properties and biofilm formation¹⁶. In the present study was designed to evaluate Aloe vera (AV) gel as a natural, oxygen-barrier post-processing medium for 3D-printed dental resins versus no-AV control, with the primary objective of determining whether AV improves physicochemical outcomes (color stability and water solubility) and a secondary, exploratory objective of examining morphology, elemental composition, biofilm formation. The null hypothesis (H₀) states that AV post-processing does not significantly alter discoloration, water sorption, solubility, morphology, elemental composition, and biofilm formation properties compared to untreated samples.

Results

Tables 1 and 2 show the time-dependent variations in ΔE00 values for Group C and Group AV. Significant differences were noted within the coffee and red wine groups in the two groups (p < 0.001, p < 0.001, respectively; Fig. 1). Furthermore, significant disparities were observed among groups in comparisons between T0–T1, T0–T7, and T0–T30 (Fig. 1). Table 3 compares the changes over time in color alteration values between Group C and Group AV in different solutions. In the coffee solution, a statistically significant difference between groups was detected solely during the T0–T30 time interval (p < 0.001). Conversely, in the red wine group, a statistically significant difference between groups was noted across all time intervals (p < 0.001) (Fig. 1).

Table 1.

Descriptive statistics and posthoc comparisons of the Group C color assessment results.

Group C	T0–T1		T0–T7		T0–T30		p
	Mean ± SD	Median	Mean ± SD	Median	Mean ± SD	Median
Distilled water	1.85 ± 0.863	1.73a	2.04 ± 1.11	1.82d	2.33 ± 0.652	2.49g	.121
Cola	2.40 ± 1.33	2.05a	1.46 ± 0.713	1.29d	2.17 ± 1.39	1.97g	.110
Coffee	5.59 ± 1.70	5.55A,b	14.2 ± 4.32	13.6B,e	18.2 ± 2.08	17.9C,h	< .001
Red wine	9.23 ± 0.977	9.43D,c	22.00 ± 3.93	23.6E,f	31.9 ± 2.62	31.7F,i	< .001
p	< .001		< .001		< .001

Group C: Post-polymerization without aloe vera gel. The p value corresponds to the Kruskal–Wallis test. The level of statistical significance was accepted as p < .05. Max: Maximum; Min: Minimum; SD: Standard Deviation; T0, Initial; T1, 1st day; T7, 7th day; T30, 30th day. Different letters show statistical differences (Uppercases indicate intra-group differences, and lowercases indicate inter-group differences).

Significant values are in bold.

Table 2.

Descriptive statistics and posthoc comparisons of the Group AV color assessment results.

Group AV	T0–T1		T0–T7		T0–T30		p
	Mean ± SD	Median	Mean ± SD	Median	Mean ± SD	Median
Distilled water	2.84 ± 1.32	2.77a	1.74 ± 1.12	1.54c	2.68 ± 2.19	1.77e	.167
Cola	2.29 ± 1.30	2.23a	2.65 ± 1.75	2.04c	2.05 ± 1.23	1.78e	.821
Coffee	5.96 ± 3.31	4.08A,b	13.6 ± 5.21	11.9B,d	14.1 ± 2.77	13.6B,f	< .001
Red wine	5.78 ± 2.84	6.20C,b	10.1 ± 3.88	10.2D,d	15.3 ± 4.55	15.4E,f	< .001
p	< .001		< .001		< .001

Group AV: Post-polymerization within aloe vera gel. The p value corresponds to the Kruskal–Wallis test. The level of statistical significance was accepted as p < .05. Max: Maximum; Min: Minimum; SD: Standard Deviation; T0, Initial; T1, 1st day; T7, 7th day; T30, 30th day. Different letters show statistical differences (Uppercases indicate intra-group differences, and lowercases indicate inter-group differences).

Significant values are in bold.

Fig. 1.

The images of samples polymerized within and without aloe vera and treated with different solutions at day 0 and day 30.

Table 3.

Descriptive statistics and posthoc comparisons of the Group C and AV color assessment results.

		Distilled water	Cola	Coffee	Red wine
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
T0–T1	Group AV	2.84 ± 1.32	2.29 ± 1.30	5.96 ± 3.31	5.78 ± 2.84
	Group C	1.85 ± 0.863	2.40 ± 1.33	5.59 ± 1.70	9.23 ± 0.977
	p	.033	.885	.272	< .001
T0–T7	Group AV	1.74 ± 1.12	2.65 ± 1.75	13.6 ± 5.21	10.1 ± 3.88
	Group C	2.04 ± 1.11	1.46 ± 0.713	14.2 ± 4.32	22.00 ± 3.93
	p	.290	.062	.694	< .001
T0–T30	Group AV	2.68 ± 2.19	2.05 ± 1.23	14.1 ± 2.77	15.3 ± 4.55
	Group C	2.33 ± 0.652	2.17 ± 1.39	18.2 ± 2.08	31.9 ± 2.62
	p	.213	.852	< .001	< .001

Group C: Post-polymerization without aloe vera gel. Group AV: Post-polymerization within aloe vera gel. The level of statistical significance was accepted as p < .05. SD: Standard Deviation; T0, Initial; T1, 1st day; T7, 7th day; T30, 30th day.

Significant values are in bold.

According to SEM analysis (Fig. 2), Group C and Group AV have presented different patterns in morphology. Group AV revealed a microtextured surface structure (Fig. 2). Figure 3 represents the EDX spectrum of Group C and Group AV. In Group C, the obtained peaks from the EDX spectrum show %wt corresponding to carbon, oxygen, nitrogen, calcium, sodium, and silisium (Fig. 3a). Figure 3b presents a higher %wt percentage of oxygen and nitrogen due to water and amino acid content of AV. It also has sodium element content due to the presence of minerals in aloe vera. The peak at 2.2 keV presents gold element response to obtain a conductive surface (Fig. 3b).

Fig. 2.

Morphological characterization of the printed (a, b) Printed samples without aloe vera and (c, d) Aloe vera-treated rinted samples at 1.000 KX and 5000 KX magnitudes.

Fig. 3.

(a) Samples without aloe vera, energy dispersive X-ray diagram showing wt% of carbon, oxygen, nitrogen, calcium, and silisium. (b) Samples within aloe vera, energy dispersive X-ray diagram showing wt% of carbon, oxygen, nitrogen, sodium, and silisium.

The data pertaining to and for Group C and Group AV are delineated in Table 4. The statistical analyses conducted did not indicate a significant difference in the values between Group C and Group AV. Group AV were found to have significantly lower levels statistically. Live bacteria were counted in the biofilm on acrylic, Group C, and Group AV. Although a statistically significant difference was not detected among the groups, Group C (1.3 × 10⁶) revealed the highest count of S. mutans. Group AV (0.5 × 10⁶) revealed similar results with the acrylic control group (0.2 × 10⁶) in regard to alive microorganism count assessments (Fig. 4).

Table 4.

Descriptive statistics and posthoc comparisons of the Group C and AV Water Sorption and Solubility results.

Groups	Group AV				GROUP C				p
	Mean ± SD	Min–Max	25–75%	Median	Mean ± SD	Min–Max	25–75%	Median
WSP	24.4 ± 12.5	10.2–47.5	19.5–24.6	22.1	24.1 ± 2.0	22.1–27.2	22.5–25.1	23.8	0.957
WSL	1.42 ± 0.69	0.0–1.7	1.7–1.7	1.7	5.1 ± 3.72	0.0–10.2	2.55–6.80	5.95	0.039

Group C: Post-polymerization without aloe vera gel. Group AV: Post-polymerization within aloe vera gel. The level of statistical significance was accepted as p < .05. Max: Maximum; Min: Minimum; SD: Standard Deviation. WSP: Water Sorption, WSL: Water Solubility.

Significant values are in bold.

Fig. 4.

Colony counting S. Mutans (CFU/mL) log10 for the evaluated resins. Group C: Post-polymerization without Aloe vera gel. Group AV: Post-polymerization within Aloe vera gel. *The level of statistical significance was accepted as p < .05.

Discussion

In the present study, samples produced with biocompatible resin have been polymerized within AV, and the impact of these attempts on discoloration, water sorption, solubility, morphology, elemental composition, and biofilm formation has been assessed. Group AV exhibited a statistically significant reduction in color changes when exposed to solutions of wine and coffee. SEM analysis revealed that Group AV exhibited a microtextured surface structure. Group AV also exhibited similar values for water sorption and a reduced rate of solubility. Although there were no significant statistical differences observed in biofilm development, a lower number of bacterial reproductions were found Group AV than Group C. Although Aloe vera contains bioactive constituents, the absence of a statistically significant antibiofilm effect in our setting likely reflects limited availability of actives after post-curing and the subsequent solvent rinse. The improvements observed in color stability and water solubility are therefore more plausibly attributable to suppression of the oxygen-inhibition layer and a higher degree of conversion, rather than to persistent surface residues. These data suggest that AV’s role in this context is primarily physicochemical rather than antimicrobial.

The results of the present study demonstrated that AV improved color stability, particularly in red wine, where discoloration was significantly lower at all time intervals. In coffee, the effect was observed only after 30 days. The stronger protective effect in wine may be due to its anthocyanin content, which binds strongly to polymeric surfaces, whereas coffee-induced discoloration results from gradual tannin accumulation, explaining the delayed effect. Haynie et al. reported high discoloration rates in 3D-printed resin brackets and found that the color change rate was high, indicating that the use of brackets made from 3D resin is not suitable in terms of color stability¹⁷. This issue is a potential aesthetic drawback in orthodontic appliances requiring prolonged intraoral use. Therefore, the removal of the OIL is crucial in maintaining color stability and ensuring the long-term aesthetic acceptability of 3D-printed dental materials. In another study, Lee et al. stated that the color durability of resins polymerized in a low-oxygen environment using glycerin was significantly higher⁵. Consistently, the present study highlights the role of oxidation inhibition in preserving color stability, emphasizing the importance of post-polymerization conditions for the longevity of 3D-printed dental materials.

AV treatment may cause a microtextured surface structure and increased flocculation on the printed materials, likely due to the high-water content of the gel, which may have partially evaporated during post-polymerization, leading to the formation of microvoids. The microtextured structure observed in Group AV may also result from residual AV remaining on the surface even after cleaning. AV’s polysaccharides, amino acids, and other bioactive components may have interfered with the polymerization kinetics, leading to a less uniform cross-linking of the resin matrix. Another possible factor contributing to the observed surface porosity is the OIL, as AV could have modified the extent of oxygen exposure during curing, thereby altering the surface polymerization dynamics. During post-curing under AV gel, oxygen is not chemically ‘removed’ from the resin bulk; rather, the gel acts as a diffusion barrier that prevents atmospheric oxygen from quenching surface radicals. Continued light exposure allows surface radicals to consume residual dissolved oxygen, thereby suppressing the oxygen-inhibition layer and increasing the degree of conversion.

The present study found that Group AV exhibited lower than Group C, likely due to AV’s high molecular weight and complex polysaccharide composition, which increased while limiting . Despite its hydrophilic nature, AV did not significantly alter , suggesting a balanced interaction with water molecules. Additionally, AV reduced oxygen permeability, potentially lowering OIL and enhancing material properties, contributing to decreased . This improvement supports moisture retention and material integrity, offering benefits for biomedical applications. In orthodontics, enhanced in 3D-printed materials may improve durability for long-term intraoral use, making AV post-processing a promising approach for stabilization splints and removable appliances. The measurement of oxygen permeability was not a direct objective of the study; therefore, the barrier effect of AV was inferred from the physicochemical outcomes rather than verified. Subsequent research endeavors should aim to quantify the oxygen transmission and the degree of conversion under AV post-curing conditions.

The incorporation of antibacterial and antibiofilm materials aids in caries prevention by limiting biofilm formation. In the study, biofilm counts did not differ significantly between groups. While a small, non-significant tendency toward lower counts was observed with AV post-curing, we do not infer an antibacterial effect under the present protocol. Any such tendency could relate to limited availability of AV components (e.g., acemannan, anthraquinones) and/or suppression of the oxygen-inhibition layer during post-curing, which might modify surface characteristics relevant to adhesion; however, this remains speculative. Prior studies similarly found no significant differences between AV and fluoride-containing agents in biofilm inhibition, suggesting that AV’s efficacy may depend on concentration and exposure time^18–20. Further research is needed to optimize its antimicrobial application.

The application of AV during the post-curing phase is hypothesized to suppress the oxygen-inhibition layer, thereby enhancing the degree of conversion and reducing the number of sorption/interaction sites. This is consistent with the observed reduction in WSL. The potential of antioxidant constituents to impede oxidative discoloration is acknowledged; however, this effect is anticipated to be negligible within the parameters of the established protocol. The color benefit is primarily physicochemical in nature. In clinical workflows, external surfaces are often finished/polished; however, the intaglio surface that primarily contacts oral tissues typically remains unpolished. Because polishing can independently alter surface roughness/energy and thereby affect staining, sorption and bacterial adhesion, we elected to keep specimens unpolished to isolate the post-processing effect. Future work should directly compare AV post-curing with vs without standardized polishing.

Although Aloe vera is known for its antimicrobial and antibiofilm properties, no statistically significant antibacterial effect was observed in the present study. This outcome may be attributed to the limited diffusion or reduced concentration of Aloe vera’s active compounds after post-polymerization, as well as the possible removal of residual components during specimen cleaning. Furthermore, the in vitro design of this study may not fully replicate intraoral conditions, including the continuous replenishment of biofluids, mechanical stresses, and dietary variations, which could influence the antibacterial efficacy of Aloe vera. Therefore, while the physicochemical benefits of Aloe vera post-processing are evident, further long-term and in vivo investigations are warranted to validate its antimicrobial potential in clinical settings.

The present study demonstrated that AV enhanced the color stability and reduced the solubility of 3D-printed dental resins, leading to the rejection of the null hypothesis in these aspects. However, while it exhibited a trend toward improved antimicrobial properties, the differences were not statistically significant, supporting the null hypothesis for biofilm reduction. These findings suggest that AV post-processing may improve the durability and aesthetics of 3D-printed dental appliances, particularly for orthodontic devices requiring extended intraoral use. Yet, intraoral conditions, including temperature fluctuations, diet, and oral hygiene, may affect these modifications over time. No mechanical finishing/polishing was performed; the results reflect unpolished surfaces. Additionally, AV may cause allergic reactions in some individuals, highlighting the need for patient history assessment before clinical use. Moreover, potential residual Aloe vera components remaining on the material surface following post-processing could have influenced the observed outcomes and should be considered when interpreting the results. Future studies should focus on in-situ and in-vivo evaluations to refine post-processing protocols and assess their clinical viability in orthodontic applications.

In addition to the aforementioned limitations, this study also has other limitations, including its in vitro design, the use of a single printable resin, and the evaluation of only one Aloe vera post-curing protocol, which may limit the generalizability of the findings. The biofilm model employed a single bacterial strain under static conditions, and staining was assessed using a fixed set of media over 30 days. Moreover, SEM/EDX analyses were qualitative and performed on representative specimens, and the study was not designed as a direct comparison with alternative oxygen-inhibition barrier materials. Finally, no clinical outcomes were evaluated, and in vivo conditions such as salivary dynamics, mechanical loading, and oral hygiene practices were not simulated.

Methods

Since no human or animal samples were used, ethical approval was not required. An a priori sample size calculation was performed in G*Power (Franz Faul, Universität Kiel, Germany) with the following settings: test family t tests; statistical test Means-Difference between two independent means (two groups); two-tailed; effect size Cohen’s d = 0.40 (medium); α = 0.05; power (1–β) = 0.90. This yielded n = 16 per group for between-group comparisons. Based on this, a total of 224 samples were initially planned (Supp Table).

The study comprised four experimental stages using disk-shaped 3D-printed specimens: (i) color stability (ΔE00), (ii) water sorption and solubility (WSP/WSL; ISO 4049:2019), (iii) surface characterization (SEM/EDX), and (iv) biofilm formation (Streptococcus mutans ATCC 25,175). Disk dimensions were 15 × 2 mm for color stability, 15 × 1 mm for WSP/WSL and SEM/EDX, and 10 × 3 mm for biofilm assays.

• Color stability n = 128 in total (Group AV = 64; Group C = 64), each group subdivided into four staining media (distilled water, cola, coffee, red wine) with n = 16 per medium; measurements were performed at T0, T1, T7, and T30.

• WSP/WSL (ISO 4049): n = 32 in total (Group AV = 16; Group C = 16).

• SEM/EDX qualitative analysis was conducted on group-representative disks (gold-coated; imaged at 1000 × and 5000 × ; EDX with Bruker XFlash 4010).

• Biofilm formation n = 32 in total (Group AV = 16; Group C = 16), assessed against S. mutans (ATCC 25175).

Disks were designed in Autodesk Meshmixer (v3.5.35; Autodesk, Inc.). STL files were oriented vertically on the production surface with support structures. Samples were fabricated using a Formlabs SLA 3D printer with Biomed Durable Clear Resin (Formlabs, Somerville, MA, USA) at a 50 µm layer thickness.

Following production, all samples were washed in 99% isopropyl alcohol for 20 min (Form Wash, Formlabs, USA), dried, and placed in 112 glass Petri dishes. Group AV samples were fully covered with 100% AV (Phytoil®, Turkey), while control samples underwent no additional treatment. Polymerization was performed at 60 °C for 60 min (Form Cure, Formlabs, USA). Afterward, all samples were rinsed with 99% isopropyl alcohol for consistent cleaning and removal of residual AV. However, potential retention of Aloe vera-derived components on the specimen surfaces cannot be completely ruled out. Finally, samples were dried using compressed air before analysis. Specimens were intentionally left unpolished to (i) mimic the clinically relevant intaglio surface that contacts teeth and mucosa, and (ii) avoid introducing finishing-related variability in surface roughness/energy.

64 samples were assigned to the Aloe vera-treated group (Group AV) and 64 to the non-treated control group (Group C), then randomly divided into four experimental groups (n = 16 per group). Samples were immersed in staining solutions: distilled water (control), coffee (Nescafé® Classic), cola (Coca Cola®), and red wine(Kavaklıdere, Turkey). To prevent bacterial contamination, solutions were renewed daily and agitated twice. Samples were incubated in darkness at 37 °C. The average daily coffee consumption (3.2 cups) equates to approximately 24 h of exposure, and a 30-day immersion period simulates around 2.5 years of staining in vivo^21,22.

Color changes in biocompatible resin samples were measured at four-time points: initial(T0), day 1 (T1), day 7 (T7), and day 30 (T30). Measurements were performed using a colorimeter (VITA Easy Shade Advance, Germany) under D65 lighting. L*, a*, and b* values were recorded in the central regions of the samples. Each sample was measured three times, with the same researcher recording and analyzing the average values. Color differences were determined using the CIEDE2000 (∆E00) formula based on the average L*, a*, and b* values²³.

In the CIEDE2000 (ΔE00) formulation, ΔL′, ΔC′, and ΔH′ denote the differences in lightness, chroma, and hue, respectively; SL, SC, and SH are the corresponding weighting functions; and RT accounts for the hue–chroma interaction in the blue region. The terms KL, KC, and KH refer to parameters related to the experimental conditions.

The disk-shaped printed samples were analyzed using SEM at magnifications of 1000 × and 5000 × . The surface morphology of the samples was observed using a ZEISS EVO 40(Merlin, Carl Zeiss), and SEM images were obtained at an accelerating voltage range of 500 V–30 kV. As the surfaces in question were not conductive, a gold coating was applied. Energy-dispersive X-ray spectroscopy (EDX) was performed using a Bruker AXs XFlash Detector 4010 to determine the percentage of nitrogen and oxygen elements present in the printed samples²⁴.

and were evaluated according to the standards set forth in ISO 4049:2019. Disk-shaped printed samples were placed in a dark oven at 37 °C. The weight change of each material was monitored until it did not exceed 0.1 mg over 24 h (n = 16). Upon reaching a consistent weight, the initial weight () was recorded with a precision of 0.1 mg. The materials were immersed in doubly distilled water at 37 °C for one week. The weights () of samples were then determined at one-minute intervals following their removal from the water. The drying and weighing procedures of the printed samples were repeated until a stable weight () was achieved. The quantities and were determined in μg/mm³, employing the following equations, wherein V denotes the volume of the printed samples in mm³²⁵.

Acrylic-based samples from Group C and Group AV (n = 16 per group) were used. Samples were acclimated in artificial saliva for one hour²⁶. S. mutans ATCC 25175 was activated from stock cultures in BHI agar at 37 °C, 5% CO₂ for 24 h. The fresh culture was inoculated into BHI broth and incubated for 16 h to reach the log phase. The bacterial suspension was prepared in BHI broth with 2% (w/v) sucrose at 1 × 10⁶ CFU/mL. In a 96-well microplate, 200 µL of saliva was added to each well, followed by aerobic incubation at 37 °C for one hour with samples immersed. After rinsing with PBS, 200 µL of S. mutans suspension (1 × 10⁶ CFU/mL) was added. Plates were incubated at 37 °C, 5% CO₂ for 48 h for biofilm development. Samples were then transferred to Eppendorf tubes with 1 mL PBS and vortexed at 3300 rpm for 30 s with two sterile glass beads to dislodge biofilms. Bacteria were enumerated via tenfold serial dilutions plated on BHI agar, with CFUs counted after 48 h of incubation at 37 °C, 5% CO₂. Results were expressed as log₁₀ CFU/mL²⁷.

Statistical analysis

All statistical procedures were conducted utilizing the open-source statistical analysis software Jamovi (The Jamovi Project, 2022, ver. 2.3.21.0, www.jamovi.org). Normality testing via the Shapiro–Wilk test revealed that the data did not adhere to a normal distribution. The Kruskal–Wallis test was employed to assess variance between groups, with post hoc comparisons conducted using the Mann–Whitney U test. A significance threshold of p < 0.05 was applied for all analyses.

Conclusion

Group AV exhibited improved color stability in coffee and red wine solutions compared to Group C. Group AV demonstrated a microtextured surface structure. Group AV and Group C showed similar values and Group C higher water solubility values, indicating less material degradation in Group AV. Although no statistically significant differences were observed, Group AV showed a trend toward reduced biofilm formation on their surfaces compared to Group C. Overall, these results indicate that the benefits of Aloe vera are predominantly physicochemical rather than antimicrobial. Further in vivo and long-term investigations are warranted to confirm its clinical applicability and to optimize post-processing protocols.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

RD: Investigation, Resources, Data curation, Experimental analysis, Writing- original draft. KGT: Validation, formal analysis, data curation, writing-review and editing, visualization, statistical analysis. MA: Conceptualization, methodology, supervision, writing—original draft, project administration. NKB: Chemical Analysis, Writing-review and editing. MS: Biological Analysis. NU: Biological Analysis.

Data availability

The data that support the findings of this research article are available from the corresponding author, upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

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