1. Introduction
Three-dimensional printing technology has revolutionized the manufacturing industry, enabling the production of complex structures with precise control over their geometry and composition. One of the emerging areas in 3D printing is the incorporation of nano-additives into polymeric matrices, resulting in nanocomposite materials with enhanced properties [
1]. However, an essential aspect that needs to be carefully evaluated is the potential leaching of these nano-additives from the printed structures when exposed to various conditions, during their lifetime.
To fully understand the potential leachable profiles of 3D-printed devices, regulatory recommendations typically call for material characterization when requesting product approval especially when it comes to medical devices, which can be accomplished via extractable/leachable (E&L) studies [
2]. The term leachable refers to a compound that migrates from a product during its intended use while an extractable is any compound that can be obtained via solvent or thermal extraction from a product or test article under controlled laboratory conditions [
3]. As such, identified extractables are potential leachables. By generating analytical data that includes both the chemical identification of extractable components, E&L studies provide information needed to evaluate both the toxicity and exposure hazard of individual compounds [
4].
Leaching studies involve the dissolution process, which refers to the release of the nano-additives into the surrounding medium. In the case of 3D-printed nanocomposite samples, the dissolution process can be influenced by factors such as the composition of the matrix, the type and concentration of the nano-additives, as well as the characteristics of the surrounding media. To investigate the dissolution process of nano-additives from 3D-printed nanocomposites, leaching tests are conducted using artificial media relevant to the potential application conditions in the use phase and in washing [
2,
3,
4].
Leaching or materials degradation releasing nanomaterials test is important for medical devices in contact with skin and wearable devices to ensure user/patient safety. Regulatory bodies require exhaustive investigations into the leachable profile of a device to ensure patient safety [
5]. Nanomaterials commonly used in wearable electronics include flexible substrates, conductors, and transducers [
6]. Silver nanoparticles (AgNPs) have been used as an antimicrobial agent in medical devices in contact with human skin due to their improved properties and excellent antimicrobial activity [
7]. Carbon fibers (CFs) have been investigated as potential constituents of medical devices for structural fixation of bone fragments, bone substitutes, and cellular growth supports in tissue engineering [
8]. In terms of skin-inspired electronics, carbon fiber/carbon black/silicone composites have been used to fabricate stretchable e-skin that mimics the tactile sensing and mechanical behavior of human skin [
9]. Exposure to carbon fibers from medical devices can cause abrasion, similarly to glass fibers, which may cause irritation to the skin or mucous membranes if uncontrolled [
10]. Furthermore, other nanomaterials such as superparamagnetic iron oxide nanoparticles (SPIONs) have been developed as novel drug delivery vehicles and have found application in magnetic resonance imaging (MRI) and magnetic hyperthermia [
11]. Upon activation of an external magnetic field [
12] or even heating induction [
13], SPIONs can promote self-healing to wearable devices via magnetic attraction. Exposure to SPIONs could potentially lead to toxic side effects such as membrane leakage of lactate dehydrogenase, impaired mitochondrial function, and DNA damage [
14]. The toxicity of SPIONs depends on their physicochemical properties, such as size, shape, surface charge, and coating [
15].
The findings from leaching tests provide valuable insights for the assessment of nanocomposite materials’ safety and suitability for various applications, including biomedical devices, consumer products, and environmental engineering. Additionally, the results can guide the development of improved formulations and manufacturing processes to minimize or control the leaching of nano-additives, ensuring the long-term performance and safety of 3D-printed nanocomposite structures.
In this study, we present a detailed workflow of the leaching test for nano-additives in artificial sweat and water media from 3D-printed nanocomposite samples through Fused Filament Fabrication (FFF) process (
Figure 1). Artificial sweat is used to mimic the composition and pH of human sweat, while water serves as a reference medium. These media provide insights into the potential release of nano-additives under conditions that simulate real-life scenarios, such as prolonged contact with the skin or exposure to environmental water sources.
2. Materials and Methods
2.1. Fabrication of 3D-Printed Nanocomposite Coupons
FFF 3D printing is the most popular additive manufacturing technique, having gained significant popularity due to the material versatility, the minimum post processing requirements, the user-friendly experience, and the accessibility to a broad range of users. For that reason, many wearable devices have been produced utilizing FFF. A commercially available 3D printer (Raise3D Pro2 Plus from Raise3D, California, USA) was used towards the production of the nanocomposite coupons. In
Table 1, the process parameters used for each nanocomposite filament are presented. Three different nanocomposite filaments comprising of different polymer matrices were used, (i) (thermoplastic polyurethane (TPU), (ii) copolyester (TX1501), and (iii) polyamide (PA12)). The chosen additives were AgNPs, chopped carbon fibers (CCFs) and SPIONs; volume fraction was preselected, according to printability studies, while material development was held within European research project Repair3D-Recycling and Repurposing of Plastic Waste for Advanced 3D Printing Applications (Grant Agreement no. 814588). In detail, the three sample categories are:
TPU with 0.75 wt% PVP-coated AgNPs (18 nm average size);
TX1501 (Tritan Copolyester TX1501HF—Eastman) with 7.3 wt% CCFs;
PA12 with 8.6 wt% CCFs and 7.5 wt% SPIONs.
In this study, TPU and PA thermoplastic matrices were used due to their strong adhesion with textiles towards the development of 3D-printed wearable devices [
16,
17] with relevant requirements. Besides the above properties, TPU presents the adequate flexibility needed for stretchable wearable devices. Furthermore, PA offers the stiffness and the flexibility required for several sports equipment and wearable textiles. The copolyester TX1501 [
18] was selected as candidate matrix for medical device applications as widely used polymer in this sector. Due to their direct contact with the skin, the materials used in this research have to be skin friendly, thus minimizing or preventing skin irritation. Thus, AgNPs which are known for their antimicrobial properties were added in the polymeric matrix [
7]. CCFs were introduced [
8,
9,
10] in matrices since they enhance the mechanical properties of the final 3D-printed parts. Furthermore, in order to increase the durability and reliability of the final 3D-printed wearable devices, SPIONs were incorporated within the thermoplastic matrix offering self-healing properties repairing minimal damages such as scratches or cracks when external magnetic field [
12] ore even heating induction is applied [
13].
2.2. Dissolution Testing
The workflow of the leaching test involved several steps. Firstly, two 3D-printed nanocomposite samples were prepared, incorporating the desired nano-additives into the polymeric matrix. In order to evaluate the leach of nanomaterials and any other additives from the polymer matrix during use or cleaning, a series of leaching tests have been performed by immersing a series of nanocomposite samples in the testing media using a dissolution apparatus (708-DS Dissolution Apparatus, Agilent, Santa Clara, CA, USA). The leaching tests take place in two different media, (i) deionized water and (ii) artificial sweat (pH = ~2.3), developed based on ISO 9022-12:2015 by Synthetic Urine EK (Eberdingen, Germany), composed of ammonium chloride, butyric acid, and acetic acid (DIN ISO 9022-12).
Each of the 3D-printed samples were immersed in both artificial sweat and water media, allowing the potential leaching from each composite during use and washing phase, respectively. In detail, the experimental setup of the leaching test comprised 8 samples:
3 samples (one of each nanocomposite type) in deionized water;
3 samples (one of each nanocomposite type) in artificial sweat;
2 controls of the media, one of deionized water and one of artificial sweat.
The experimental setup included the position of each sample (3D-printed discs) in designated baskets immersed in 1 L of media, while the controls included the same media volume as well, all under continuous stirring (50 rpm) and 37 °C with the provision of 1 month of continuous immersion in the media. The water bath of the dissolution apparatus was set slightly higher (37.2 °C) to ensure that the temperature at each vessel was 37 °C and that the desired temperature was achieved before the experiment began. An auto-sampling device was used to continuously monitor the leaching test. A series of 12 auto-samples were taken over approximately one month using a programmed dissolution apparatus. Sample 1 was taken immediately after start (time 0), Sample 2 was taken an hour later, Samples 3–4 were collected every 2 h, Samples 5–8 were taken daily at the same time as Sample 1, and Samples 9–12 were taken weekly at the same time as Sample 1. After each set of samples, they were transferred to sterile falcon tubes and stored in a refrigerator at 4 °C until further analysis (10 mL per sample). Temperature and sampling time were recorded simultaneously. To ensure the accuracy of samples and reduce the risk of contamination, each sample was labeled and sealed with parafilm. The door of the auto-sampling device’s remained closed during the process to minimize potential contamination from airborne hazards.
2.3. SEM/EDS and UV-VIS Analysis
A UV-VIS spectrophotometer was used (V-630, Jasco Inc., Easton, MD, USA) to measure the ultraviolet and visible light absorption spectra (UV-VIS) of the collected media samples after dissolution testing. The 3D-printed components were subjected to scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) (Phenom X, Themoscientific, Waltham, MA USA) to investigate the surface morphology and elemental composition of the 3D-printed parts. Sputter coating (Quorum SC7620) with Au/Pd for 90 s at 20 mA was employed prior to SEM/EDS assessment. The deposition of the Au/Pd layer, enhances the conductivity of the polymeric samples and reduces charging effects, improving SEM image quality and EDS analysis.
2.4. In Vitro Skin Sensitizing Potential of the Materials before and after Leaching Test
Prior to in vitro testing, material samples were sterilized by being immersed in 70% ethanol (v/v) overnight, followed by rinsing three times with 1× phosphate-buffered saline (PBS) to eliminate all traces of ethanol. Then, the samples were air-dried in a sterile atmosphere before being sterilized for 1 h with ultraviolet light. This phase ensured that any pollutants on the surface were removed to avoid contamination of the cell cultures. The sterile samples were immersed in complete Dulbecco’s Modified Eagle Medium (DMEM) overnight before testing. Hs27 skin cells (human fibroblasts, skin, foreskin) complying with the ISO 10993-5 standard for cytotoxicity testing, were purchased form American Type Culture Collection (ATCC, Manassas, VA, USA) were grown to confluence in complete DMEM containing 10% FBS and 1% penicillium/streptomycin solution at 37 °C in an incubator with 5% carbon dioxide (CO2). In 6-well plates, samples were placed in each well, followed by direct cell seeding at a seeding number of 1 × 504 cells per well, and the plates were placed in the CO2 incubator. As a positive control, a tissue culture plate with fresh DMEM and cells was used. After 24 and 48 h of exposure, the release of the proinflammatory cytokines Interleukin-18 (IL-18) and Tumor Necrosis Factor-α (TNF-α) as indicators of possible skin sensitization, released in the cell culture medium was measured by Enzyme-linked Immunosorbent Assay (ELISA) (OriGene, EA100026 and CAYMAN CHEMICAL,589201, respectively), according to the manufacturer’s protocol.
3. Results
3.1. UV-VIS Analysis of the Samples after Dissolution
UV-VIS spectroscopy was utilized to investigate material degradation and leaching of CCFs and NPs during dissolution in artificial sweat composed of ammonium chloride, butyric acid, and acetic acid with a pH of approximately 2.3, and/or water for a period of 32 days. Three distinct nanocomposite filaments were used to produce 3D-printed test coupons utilizing FFF.
3.1.1. TPU with 0.75 wt% AgNPs
As shown in
Figure 2a, the UV-VIS spectroscopy analysis of TPU composites with 0.75 wt% AgNPs presents two absorption peaks at 250 nm and 320 nm. Based on the literature, AgNPs are known to exhibit a UV-VIS absorption maximum in the range of 400–500 nm [
19] because of surface plasmon resonance (SPR). However, no peak between 400 and 500 nm, characteristic of AgNPs, was observed. The SPR band is a result of the collective oscillation of conduction electrons in the silver nanoparticles when excited by incident light. It is important to note that the exact position of the peak can vary based on factors such as nanoparticle size, shape (spherical, rod-shaped, etc.), aggregation state, and capping agents [
20]. While it is possible for silver nanoparticles to exhibit some absorption at wavelengths below 380 nm, it is generally not significant. At 250 nm, the absorption by silver nanoparticles is usually very weak or negligible. Manish Kumar et al. [
21] reported that PVP exhibits maximum absorption peak at 243 nm, while PVP-coated AgNPs demonstrate maximum absorption at 406 nm. The obtained results showed two distinct peaks in the UV-VIS spectrum, one at 250 nm and another at 320 nm, which both correspond to AgNPs, indirectly and directly, respectively [
22]. The first peak at 250 nm corresponds to the presence of the PVP-coated AgNPs, interacting with other chemical species inside the bath solution, while the second peak at 320 nm corresponds to AgNPs. Prolonged exposure of the samples inside the dissolution media resulted (a) in surface deterioration of the 3D-printed specimens and (b) in the release of chemical species that influence the UV-VIS absorption. As the residence time increases in the dissolution media, the more the surface deteriorates, and the more AgNPs are released from the polymer matrix (increasing absorbance of the peak at 250 nm). Regarding the artificial sweat solution, the AgNPs released from the polymer matrix in the dissolution media, resulting in observable changes in the UV-VIS absorption. Eventually, the leaching of PVP from the AgNPs reaches a maximum level after 18 days and then disappears, probably due to nanoparticle precipitation in the artificial sweat medium, as indicated by the disappearance of the characteristic nanoparticles peak in the UV-VIS analysis at 250 nm. The UV-VIS analysis of water samples after dissolution resulted in minor but not significant observations of leaching of the nano-additives, indicating that washing conditions might not affect the 3D-printed nanocomposite material (described in
Figure 2b).
3.1.2. TX1501 with 7.3 wt% CCFs
Regarding the UV-VIS absorption of the TX1501 nanocomposite tested coupons immersed in artificial sweat, two distinct peaks are evident in 240 and 250 nm (
Figure 3a). According to the literature, polyesters present a characteristic maximum absorption peak around 240 nm [
23], while chopped carbon fibers may exhibit structure-dependent variations in their maxim UV-VIS peak absorption.The UV absorption peak in carbon materials is associated with electronic transitions between the bonding and antibonding π orbital [
24]. The π-π* transitions appear in the range of 180–260 nm in all the carbon materials, as reported in the literature [
25]. UV-VIS analysis of water samples after dissolution (
Figure 3b) resulted in minor but not significant observations of leaching of the nano-additives, indicating that washing conditions might not affect the 3D-printed nanocomposite material.
3.1.3. PA12 with 8.6 wt% sCFs and 7.5 wt% SPIONs
UV-VIS spectrum for the PA12 nanocomposite filament was obtained and is presented in
Figure 4. A characteristic absorbance peak between 250 and 260 nm is evident. Based on previous studies [
26,
27], SPIONs exhibit maximum absorbance near 250 nm. The exact position of the maximum peak, within this range, could vary due to the absorbance of the organic molecules inside the tested solution. Several studies have reported [
28,
29] the UV-visible spectroscopy wavelength of Fe
3O
4 nanoparticles to be present at 262.13 nm and 230 nm. The 262.13 nm band seems to be in agreement with our observed peaks at 260 nm, leading to the conclusion that a number of Fe
3O
4 nanoparticles might have been released from the nanocomposite PA12 material during leaching test in the artificial sweat, while the second peak at 320 nm corresponds to PA12 [
30]. As mentioned in TX1501, the π-π* transitions of the CCFs appear within the range of 180–260 nm. However, SPIONs, which consist of the inclusions in PA matrix, absorb the UV light in the range of 250–260 nm. It is possible, though, that SPIONs absorption overlaps the absorption of the CCFs due to the increased plasmonic effect; therefore, there is no clear evidence relating to CCFs release. Additionally, CCFs contained in the nanocomposite PA12 3D-printed samples detected on the surface are entrapped within the polymeric matrix, reducing the possibility of detachment and release. Even if an amount of CCFs was released, the concentration may be below the detection limit and, thus, not being detectable. The UV-VIS analysis of water samples after dissolution resulted in minor but not significant observations of leaching of the nano-additives, indicating that washing conditions might not affect the 3D-printed nanocomposite material.
3.2. SEM/EDS Assessment
The presence of inclusions within the polymeric matrix materials can have a significant impact on the overall material performance and durability during use-phase, especially when they come into contact with external environments and skin. In order to understand and evaluate the dissolution characteristics of these inclusions, a comprehensive analysis utilizing scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) techniques was conducted. These analyses include morphology and stoichiometric examination of SEM images, cross sections of raw materials, longitudinal section of the filaments used, and surfaces of 3D-printed parts, allowing us to conduct a detailed investigation into the morphology and composition of the inclusions. By connecting the surface morphology with the leaching behavior, this study aims to provide valuable insights into the potential effects of inclusions on material performance and help inform the design and selection of materials for various applications.
Initially, SEM assessment of raw nanomaterial powders was performed to evaluate their morphology and composition. It is evident that both AgNPs and SPIONs form aggregates with large size variation and abstract shape (
Figure 5). To evaluate the nanocomposite filaments used for the production of the 3D-printed test coupons, surface examination of the filaments and longitudinal filament sections were assessed through SEM (
Figure 6,
Figure 7 and
Figure 8). Despite the fact that AgNPs and SPIONs were difficult to find on the surface of the composite filaments, they were apparent within the polymer matrix. It is possible, though, that the polymer matrix or surface contaminants may obscure or mask the NPs, thus compromising the EDS analysis. The surface morphology of 3D-printed test coupons right after the fabrication process was also assessed. As previously mentioned, CCFs are evident on the surface of the 3D-printed samples either totally or partially covered by the polymer matrix. It was not possible to detect NPs through the EDS, suggesting that due to the polymer extrusion flow from the heated nozzle, the NPs are trapped into the polymer matrix (
Figure 9). Cross-section SEM images were taken from each 3D-printed test coupon. CCFs are once again evident, but the NPs were hard to determine due to possible surface contaminants (
Figure 10).
After the immersion in artificial sweat bath, the 3D-printed coupons underwent surface deterioration due to the high temperature, pH, and extended exposure time in the sweat bath. It was not possible to detect CCFs and NPs on the surface of the 3D-printed coupons, with the exception of the TX1501 sample, where CCFs were discovered. The absence of detectable NPs and CCFs on the surface of the samples after the dissolution process may lead to the following hypotheses:
- (i)
The CCFs and NPS may be leaching out in a dispersed form and have been detached from the surface. This could be possibly explained by the UV-VIS absorption experiments when PVP peaks were observed instead of AgNPs, as mentioned above.
- (ii)
Because of the low concentration of CCFs and NPs in the nanocomposite materials and due to the extrusion process while 3D printing, the added materials are trapped within the polymeric matrix, making challenging the detection of the CCFs and NPs on both the surface and cross section of the tested coupons.
- (iii)
The presence of salts on the surface of the immersed samples as well as their thickness and coverage area compromised the EDS analysis. Due to the detection limitations of the instrument, the verification of NPs on the surface of the samples immersed in artificial sweat can only be performed visually comparing their morphology directly with the raw NPs powder.
On the contrary, samples immersed in deionized water present partial surface deterioration, sustaining, however, their initial morphology. SPIONs and CCFs on the surface of the PA as well as CCFs on TX, are evident. This could be aligned with the UV analysis, suggesting that the leaching may occur in lower time rates in water than in artificial sweat (
Figure 11).
3.3. Dimensional Accuracy/Stability Assessment
Dimensional assessment analysis was performed to determine any alterations in size, while weight measurements were employed to assess any variations in mass of the 3D-printed tested coupons. Comparing the pre-dissolution and post-dissolution results, the structural integrity of the samples was examined. The measurements obtained from this study contribute to a better understanding of the dissolution process and its impact on the physical properties of the samples. No significant size or weight changes were found, indicating that the bulk properties of the 3D-printed samples were not severely affected, as shown in
Table 2, except for the total weight gain (%) of the polyamide (3.04% weight gain), attributed to the behavior of polyamide being prone to water/moisture uptake, also reported in the literature [
31].
3.4. In Vitro Skin Sensitizing Potential of the Materials before and after Leaching Test
Prior to in vitro testing, the 3D-printed samples before and after the leaching test in artificial sweat were sterilized as already described to avoid contamination in the cell cultures. Due to the fact that no significant alterations were observed after the leaching test in water, it was considered that washing conditions do not affect the material’s skin sensitizing potential.
The sterile samples were immersed in complete DMEM overnight before cell seeding containing 10% FBS and 1% penicillium/streptomycin solution at 37 °C in an incubator with 5% carbon dioxide (CO2). After 48 h of exposure of the skin cells to the materials, the release of the proinflammatory cytokines IL-18 and TNF-α as indicators of possible skin sensitization in the cell culture medium was measured by ELISA, according to the manufacturer’s protocol.
After performing the calibration curves for IL-18 and TNF-α and the subsequent analysis of concentrations (pg/mL) obtained by means of the equation of the straight line generated by the calibration curve, the average of each sample was calibrated and compared with the respective control (only cells without any material, not shown in the graph), and materials before and after leaching were compared to each other as shown in the graphs (
Figure 12). The results were analyzed by performing a one-way analysis of variance (ANOVA) test.
p-values > 0.05 were not considered statistically significant. Although TPU samples after leaching seem to minimally increase the detection of the proinflammatory cytokines, no significant changes were observed, leading to the conclusion that leaching tests in artificial sweat, simulating use conditions, might not affect the material’s skin sensitizing potential. Measurements of more inflammatory factors would better elucidate this finding.
4. Discussion
The aim of this study was to develop a comprehensive process for evaluating the leaching behavior of 3D-printed nanocomposite samples intended for use in wearable devices. The research focused on three different nanocomposite filaments, each composed of distinct polymer matrices, including TPU, TX1501, and PA12. The filaments contained various additives such AgNPs, CCFs, and SPIONs. The investigation utilized UV-VIS spectroscopy to assess the leaching of nano-additives in artificial sweat, which showed a gradual leaching followed by a plateau. The UV-VIS analysis of water samples after dissolution resulted in minor but not significant observations, leading to the conclusion that no leaching of the nano-additives was present, indicating that washing conditions might not affect the 3D-printed nanocomposite material. To gain insights into the morphology and composition of the inclusions, SEM and EDS were employed. SEM analysis was conducted on the raw nanomaterial powders, nanocomposite filaments, and the 3D-printed test coupons. The examination revealed that AgNPs and SPIONs formed aggregates with variable sizes and abstract shapes. While AgNPs and SPIONs were not easily detectable on the surface of the composite filaments, they were present within the polymer matrix. The presence of CCFs was observed on the surface of the 3D-printed samples, either partially or entirely covered by the polymer matrix. However, the NPs were challenging to detect using EDS analysis, possibly due to their entrapment within the polymer matrix during the extrusion process. After immersion in artificial sweat, the 3D-printed coupons exhibited surface deterioration, which made it difficult to detect CCFs and NPs on their surfaces, except for the TX1501 sample, where CCFs were found. Several hypotheses were proposed to explain the absence of detectable CCFs and NPs on the surface after the dissolution process. These included the possibility of dispersed leaching, where CCFs and NPs detached from the surface, or the trapping of added materials within the polymer matrix during 3D printing, making their detection challenging. The presence of salts on the surface of the immersed samples, as well as their thickness and coverage area, could have also affected the EDS analysis. Immersion in deionized water resulted in partial surface deterioration, maintaining the initial morphology, and the presence of SPIONs and CCFs on the surface of PA12 and CCFs on TX1501. Furthermore, the weight and dimensions of the 3D-printed samples did not exhibit significant changes before and after the dissolution process, indicating the stability of the materials during the testing. An exception to this is the total gain weight of PA12. Prior to in vitro testing, the samples underwent sterilization to eliminate surface contaminants, ensuring the absence of contamination in cell cultures. The cytotoxicity of the samples was evaluated using human fibroblast cells, and the release of proinflammatory cytokines IL-18 and TNF-α was measured. Calibration curves were generated, and the concentrations of cytokines were compared to respective controls. Statistical analysis revealed no statistically significant differences (p > 0.05). Overall, this research provides valuable insights into the leaching behavior and morphology of inclusions in 3D-printed nanocomposite samples. The study highlights the challenges in detecting nano-additives on the surface and within the polymer matrix of 3D-printed components. The results contribute to the understanding of material performance and help inform the design and selection of materials for wearable technologies, facilitating their safe and reliable use in various applications.
5. Conclusions
In conclusion, this study aimed to develop a comprehensive process for evaluating the leaching behavior of 3D-printed nanocomposite samples intended for devices. Through materials characterization, in vitro toxicity testing, and analysis of leaching behavior, several key findings were obtained. The UV-VIS spectroscopy results demonstrated the leaching of nano-additives in artificial sweat, reaching a plateau over time. SEM and EDS analyses provided insights into the morphology and composition of inclusions within the nanocomposite filaments and 3D-printed samples. The weight and dimensions of the 3D-printed samples did not exhibit significant changes before and after dissolution, indicating their stability during testing. In vitro toxicity testing using human fibroblast cells showed no statistically significant differences in the release of proinflammatory cytokines IL-18 and TNF-α between the samples and controls. Overall, this research contributes to the understanding of leaching behavior, morphology, and composition of 3D-printed nanocomposite samples. The findings support the development of safe and reliable 3D-printed materials for wearable technologies, facilitating their adoption in various applications. Future studies could explore additional characterization techniques and optimize the detection of nano-additives in 3D-printed materials to further enhance their performance and safety.