Hidden cytotoxicity and mitochondrial dysfunction in 3D-printing polymers: evidence from FLEX, PETG and PC

Hidden cytotoxicity and mitochondrial dysfunction in 3D-printing polymers: evidence from FLEX, PETG and PC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hidden cytotoxicity and mitochondrial dysfunction in 3D-printing polymers: evidence from FLEX, PETG and PC Jiří Dejmek, Jan Jedlička This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8095943/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Additive manufacturing, also known as 3D printing, is a rapidly evolving technology that is profoundly impacting consumer products and biomedical applications. The persistent lack of essential toxicological data in material safety data sheets (MSDS) for additive manufacturing raises legitimate concerns regarding the biological safety of the polymers utilized in 3D printing. In this study, the cytotoxic potential of eight widely available filaments—polylactic acid (PLA), polyethylene terephthalate (PETG), chlorinated polyethylene (CPE), polycarbonate (PC), acrylonitrile butadiene styrene (ABS), polypropylene (PP), and flexible polyurethane (FLEX)—was examined using an ISO 10993-5 compliant indirect contact assay on primary human dermal fibroblasts. Cells were exposed to leachables diffusing from 3D-printed inserts for 24 hours or 7 days, and viability, proliferation, metabolic activity, and mitochondrial respiration were assessed. The investigation revealed that FLEX (thermoplastic polyurethane), PETG, and PC induced significant cytotoxic effects, including impaired proliferation, altered morphology, and disrupted mitochondrial respiration. Conversely, PLA, ABS, and CPE demonstrated minimal impact under the tested conditions. The observed toxicity is likely associated with additives, pigments, and plasticizers, such as isocyanates or volatile organic compounds (VOCs). These compounds are released during the thermal degradation of the material during printing. Specifically, the toxicity profile aligns with the known hazards of residual isocyanates in FLEX, glycol modifications in PETG, and the known release of bisphenol A and related compounds from PC. These findings suggest that materials commonly regarded as biocompatible may exhibit hidden toxicity due to additives or degradation by-products generated during the printing process. The findings of this study underscore the imperative for a systematic toxicological evaluation and stringent regulatory oversight of 3D-printing polymers, particularly given their pervasive use in consumer contact applications—including wearables (such as customized shoes and wristbands) and items intended for vulnerable populations (such as infant and toddler toys)—where direct and long-term exposure indicates a potential, yet unrecognized, risk to public health. Physical sciences/Chemistry Physical sciences/Materials science 3D print cytotoxicity Filament PLA PET ABS ASA PC PP TPU CPE FLEX fibroblast cell viability mitochondria respirometry Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Additive manufacturing, also known as 3D printing, has emerged as a prominent advanced technology that is fundamentally transforming the production of objects and structures across a wide range of applications, from rapid prototyping to the production of biomedical implants. Its application has proliferated across numerous sectors, encompassing domains such as healthcare, education, and sports. A particular and expanding category is that of wearable devices, which have evolved into a pivotal component of electronics (e.g., bracelets), textile technologies, and footwear design. A wide range of polymer materials in the form of filaments, such as PLA, PETG, CPE, ABS, and FLEX, are used for their production. These materials are available in a variety of colors and possess specific chemical properties that provide a high degree of flexibility in design and potential applications. A comprehensive study of recent advances in the additive manufacturing of smart textiles was conducted by (Manaia et al., 2023 ). The research is centered on the domains of 3D and 4D printing technologies, with a focus on their potential for the development of future multifunctional textiles. (Song et al., 2023 ) presented a microfluidic diagnostic wearable device, created using 3D printing and integrating smart microfibers for medical purposes. This study highlights the potential of microfluidic systems for health diagnosis and monitoring. Given the expanding utilization of additive manufacturing for the fabrication of consumer goods and biomedical applications, it is imperative to evaluate the potential health risks stemming from the interaction of these polymer materials with biological systems. Specifically, the cytotoxic effects on human cells have not yet been sufficiently studied. A comprehensive evaluation of cellular toxicity is imperative to guarantee the safe and effective utilization of these materials, particularly in the context of direct or indirect contact with the human body (Derby, 2012 ; Murphy & Atala, 2014 ; Tetsuka & Shin, 2020 ). In their publication, (Tetsuka & Shin, 2020 ) critically reviewed the current trends in 3D-printed medical wearable devices and addressed potential health risks. Key concerns include the possibility of allergic reactions to printing materials and insufficient sterility of prints, which can lead to infection or implant rejection, compromising the safety and effectiveness of treatment (Augustine et al., 2025 ; He et al., 2018 ; Tappa & Jammalamadaka, 2018 ). In the realm of fundamental in vitro research, where 3D printing technology is employed to engineer specialized platforms for the study of cell motility, biological materials such as scaffolds, and microfluidic systems (Augustine et al., 2025 ; Chander & Mahajan, 2024 ; Farcas et al., 2019 ; Guttridge et al., 2022 ; J. M. Lee et al., 2016 ; Tuomi et al., 2017 )knowledge of its potential cytotoxic effects is paramount. The scientific literature contains ample documentation of the toxicity of a range of polymer materials (Manoochehri et al., 2024 ; Zimmermann et al., 2019 ). Notwithstanding this awareness, the dearth of crucial toxicological data in material safety data sheets (MSDSs) for additive manufacturing engenders valid concerns regarding their biological safety. Published data on the assessment of the material's cytotoxic properties for 3D printing applications are currently very limited, especially with regard to their filament form. Furthermore, filament manufacturers typically do not provide biocompatibility assessments in accordance with relevant standards, such as ISO 10993-5. These gaps in knowledge underscore the necessity for further systematic research. Research conducted by (Guttridge et al., 2022 ) and (Satzer & Achleitner, 2022 ) has previously indicated that the utilization of prevalent materials and their surface chemical composition may exert an influence on cell viability, proliferation, and morphology. These findings underscore the pivotal function of material-cell interaction in ensuring the reliability and efficacy of cell experiments in biomedicine and bioengineering. The toxicity of color pigments has been demonstrated to pose a potential risk, thereby further complicating the safety assessment of finished products (Chia & Wu, 2015 ; Mota et al., 2015 ; Tetsuka & Shin, 2020 ). A paucity of comprehensive information exists regarding the cytotoxic potential of materials commonly utilized in three-dimensional (3D) printing applications. In light of this deficit, the present study was conceived as a preliminary evaluative investigation of sixteen distinct polymers. The evaluation encompassed simulated scenarios of indirect contact, such as the utilization of 3D-printed wearable devices or footwear, or the employment of 3D-printed devices and inserts in the context of cell research. The following working hypothesis was formulated: the exposure of primary human dermal fibroblasts (HDF) to indirect contact with custom-designed 3D-printed inserts made from PLA, CPE, PETG, ABS, ASA, PC, PP, and FLEX will have a negative influence on their viability, proliferation, metabolic activity, and mitochondrial respiration (Gnaiger, 2014 ; S. E. Lee et al., 2022 ; Präbst et al., 2017 ; Salin et al., 2015 ). The degree of toxicity will depend on both the type of material and the duration of exposure (short-term, 24 hours; long-term, 7 days). HDF cells were selected as a robust mesenchymal model system that is highly relevant for evaluating human skin contact with plastics. The assessment of cellular toxicity was conducted in accordance with the established principles of ISO 10993-5:2009/A11:2025, employing an indirect contact method that ensured cells were not in direct physical contact with the polymer specimens. Instead, cells were continuously exposed to potential leachables that were diffusing from the material into the culture medium. In accordance with the ISO 10993-5 criteria, a reduction in cell viability of ≥ 30% relative to the negative control was considered indicative of toxicity. The efficacy of indirect contact via inserts has been previously validated for a range of material types, including dental biomaterials (Babich et al., 2009 ), crosslinked polymers (M. O. Wang et al., 2013 ), and 3D-printed constructs (Rengarajan et al., 2023 ). Therefore, it was deemed suitable for the present study to reproduce realistic scenarios of continuous leaching without direct material–cell contact. MATERIALS AND METHODS 3D print filaments (materials) For the purpose of this study, a total of 16 different types of commercially available 3D printing filaments were selected to test the hypothesis. The selection criteria encompassed a range of polymer types, along with various color variants of these polymers. A comprehensive list of the tested filaments, accompanied by their respective manufacturers, is provided in Table 1 . Each sample was assigned a unique identifier in the first column of the table to facilitate reference and clarity in the presentation of the results. This structured approach ensures traceability and proper identification throughout the analysis. Table 1 – Table of polymer filaments used in this study. Material ID column serves for identification of material trough experiment and this publication. Column polymer express filament polymer material. Filament type express producer additional identification. Printed temperature in [°C] represent overall print temperature and heated temperature. Producer column represents filament producer.. Material ID Polymer Filament type Print temperature Producer F1 PLA Extrafill Crystal Clear 220/60 Fillamentum F2 PLA Extrafill Trafic White 220/60 Fillamentum F3 PLA Extrafill Trafic Black 220/60 Fillamentum F4 PLA Extrafill Natural 220/60 Fillamentum F5 CPE HG100 Extrafill Natural 270/90 Fillamentum F6 ABS Extrafill Black 240/110 Fillamentum F7 ASA Extrafill Natural 260/110 Fillamentum F8 ABS Extrafill Natural 240/110 Fillamentum F9 ABS Extrafill Transparent 240/110 Fillamentum F10 FLEX A98* Trafic White 240/50 Fillamentum F11 PC-ABS Natural 270/110 Fillamentum P1 PLA Jet Black 225/60 Prusament P2 PETG PETG Jet Black 250/90 Prusament P3 PC Blend 275/115 Prusament S1 ABS MEDICAL 240/100 Smartfill V1 PP PP transparent 240/80 Verbatim Test sample preparation For the in vitro testing, a custom-designed insert was fabricated. The dimensions of the specimen are presented in millimeters and illustrated in Fig. 1 B. The insert consists of eight cylindrical hollow wells, precisely sized to fit into a single row of a 96-well plate. The height of the insert was designed to be less than the depth of the well to prevent direct contact between the tested polymeric material and the cellular components. The dimensions and medium contact surface of the inserts are identical for each material that was tested. The three-dimensional model of the insert was created using SolidWorks software (Dassault Systèmes, S.A.). The 3D model was subsequently prepared for printing using PrusaSlicer v2.4.1 software (Prusa3D, CZ). The inserts were fabricated using a standard, commercially available Prusa MK3s 3D printer (Prusa3D, CZ). In order to mitigate the risk of contamination from airborne particles, viruses, and bacteria, the printers were encased in a hermetically sealed enclosure. To assess bacterial sterility, ten samples were selected at random and placed into microbial cultivation broth after the completion of the 3D printing process. The samples were then incubated at 37°C, 100% relative humidity, and 5% carbon dioxide for a period of seven days. Subsequently, each sample was examined both macroscopically (for turbidity) and microscopically (for the presence of bacteria). No contamination was observed in any of the test samples. All the inserts were aseptically preserved without undergoing any additional sterilization until their utilization in the experiment. Chemicals In the absence of an explicit statement to the contrary, all chemicals, assays, and cell culture plastics were procured from VWR International, a subsidiary of Avanton, located in Prague, Czech Republic. Devices The immunofluorescence assessment of cells and their respective extracts was conducted using the Cytation5 plate reader (Agilent, USA) instrument with Gen5 software (version 3.10, Agilent, USA). Cell culture images were captured using an inverted microscope (Olympus IX75) with a Canon EOS 1300D camera and QuickPhoto PRO software (version 3.2 build 1887-II, Promedika, CZ). The measurement of mitochondrial respiration was performed on Oroboros O2k oxygraphs (Oroboros, Austria). Cell culture and precultivation The cell line of human dermal fibroblasts (HDF-ax3027) was obtained from Axol Bioscience (Easter Bush Hub, UK). HDF were cultivated in DMEM Medium (Biowest, L0101-500) with the following supplements: 10% (v/v) fetal bovine serum (Merck, F7524), 1% (v/v) penicillin-streptomycin (Merck, P4333), and 2.5 mmol/L L-glutamine (Merck, G7513). The cultivation took place at 37°C under 5% CO2 in a humidified incubator. For subculturing, cells were washed with Dulbecco phosphate-buffered saline (Merck, D8537) and then briefly incubated with TripLE Express (Gipco, 12605-028). Experiment protocol The assessment of cellular toxicity was conducted in accordance with the established principles of ISO 10993-5:2009/A11:2025, employing an indirect contact method that ensured cells were not in direct physical contact with the polymer specimens. Instead, cells were continuously exposed to potential leachables that were diffusing from the material into the culture medium. To emulate acute exposure, cells were exposed to samples of the tested materials for a period of 24 hours. The simulation of chronic exposure was achieved by subjecting cells to samples of the tested materials for a period of seven days. The HDF cells were meticulously dispensed into 96-well plates (Avantor, model 734–2327) with a density of 5 x 10³ cells per well. Throughout the experiment, the cells were cultivated under standard conditions in an incubator (air + 5% CO₂). Subsequently, on the following day, 16 wells (N) were prepared for each material, and 3D-printed inserts were placed into these wells. The inserts were composed of the selected materials and were designed to be placed into wells containing planted cells, as illustrated in Fig. 1 (D). Each plate also contained a control group of unaffected cells (N = 16). The culturing medium was changed on the fourth day of the experiment. For the HRR method (chronic exposure only), cells were cultured for seven days in a 250-ml bottle (Avantor, 734–2809) in the same culture medium and under the same conditions. Cell morphology The evaluation of cellular morphology was conducted through optical comparison, employing visual analysis of cellular structures under a microscope with appropriate magnification and illumination. In summary, images of cell culture were obtained on the second (acute) and seventh (chronic) days of the experiment. The subsequent morphological parameters between the control and affected cells will undergo comparison. The dimensions of cells have the potential to serve as indicators of alterations in the cytoskeleton, adhesion processes, or the cell cycle. The presence of vacuoles, inclusions, or lipid droplets in the cytoplasm of affected cells may be indicative of changes in metabolic activity or the presence of pathological changes. The degree of homogeneity in cell growth and the pattern of cell growth can provide information about proliferation, adhesion, and cell communication. Cell proliferation To assess and compare the growth of treated and control cells, live cell nucleus staining with NucBlue® (Invitrogen, Life Technologies, Prague, CZ) was utilized. On the second and seventh days of the experiment, filament sample inserts were extracted from the culture plates. Subsequently, the medium was aspirated, and the cells were rinsed with phosphate-buffered saline (PBS), which was also aspirated. Subsequently, 100 µl of the LCIM solution containing NucBlue at the manufacturer's recommended concentration (i.e., two drops per 1 ml of media) was added to the wells. The cells were then subjected to a culture process in an incubator maintained at a temperature of 37°C in an environment containing 5% CO2 for a duration of 20 minutes. Subsequently, the darkened samples were inserted into the Cytation5, and the number of nuclei in each well was precisely determined with a sample or control using the recommended protocol (λex = 360 nm; λem = 460 nm) of the Gen5 software. Single cell fluorescence (SCF) is defined as the average intensity of the fluorescence signal from a single cell. That is to say, it is the ability of NucBlue stain to penetrate the cell nucleus and intercalate into the DNA. It is also indicative of the presence of cell nucleus morphology abnormalities. The value of SCF was determined by the ratio of the absolute number of cells in a well to the total fluorescence response (FU) of a given well. The resultant value is designated as the SCF value. Cell Viability To compare the metabolic activity of the treated and control cells, the PrestoBlue® fluorescent reagent (Invitrogen, Life Technologies, Prague, CZ) was employed. PrestoBlue is a rapid cell viability indicator that utilizes the reducing power of live cells to convert resazurin to the fluorescent molecule, resorufin (O'Brien, Leiphrakpam, Xiao). The value of the absorption of the fluid above the precipitate, which represents the current concentration of blue resazurin and metabolized pink resorufin, was calculated according to the protocol established by the manufacturer, as detailed in the product documentation. The resulting value, denoted in the results as Single Cell PrestoBlue reduction rate (SCPB), represents the ratio of reduction capacity between the treated and control cells and is normalized per cell. On the second and seventh days, the inserts from the culture plates were removed. Subsequently, the medium was aspirated, and the cells were rinsed with phosphate-buffered saline (PBS). Subsequently, 100 µl of LCIM solution with PrestoBlue (at the manufacturer's recommended concentration, i.e., 100 µl per 1 ml of media) and NucBlue (at the manufacturer's recommended concentration, i.e., 2 drops per 1 ml of media) were added to the wells. The cells were then subjected to a culture process in an incubator maintained at a temperature of 37°C and a humidity level of 5% carbon dioxide for a duration of 120 minutes. Subsequently, the samples were inserted into the Cytation5, and using Gen 5 software, our protocol determined the absolute cell count in each well, the fluorescence value of the wells (FU), and the absorbance of wells with cells and wells without cells (blank). The data obtained was then utilized to calculate the results. High Resolution respirometry HRR The cells were then placed into four pre-calibrated oxygraphs (O2k, Oroboros, Austria), each containing two chambers with a volume of 2 ml at 37°C. Subsequent to the attainment of equilibrium, measurements were initiated using a standardized SUIT (Substrate-Uncoupler-Inhibitor-Titration) protocol. Subsequent to the ROUTINE state (respiration of unaffected intact cells), the following states were measured: LEAK: Respiratory compensation for a proton leak across the inner mitochondrial membrane following the inhibition of ATP synthase by oligomycin (OMY; 2.5 µmol/L, as determined by titration in a preliminary experiment). ETS: Electron Transport System Capacity, defined as maximal respiration subsequent to the uncoupling of oxidation and phosphorylation by FCCP (carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; 0.05 µmol/L, gradual titration). ROX: Residual oxygen consumption originating from sources other than the electron transport chain, titrated using rotenone (ROT; inhibitor of complex I; 0.5 µmol/L) and antimycin A (AMA; inhibitor of complex III; 2.5 µmol/L). The activity of complex IV was determined by titration with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride; an artificial substrate of complex IV; 0.5 mmol/l), ascorbate (a reducing agent for high auto-oxidation of TMPD; 2 mmol/l), and azide (AZD; an inhibitor of complex IV; 100 mmol/l). The data were processed using DatLab software, version 7.4. Citrate synthase activity The medium for determining citrate synthase activity consists of the following components: 0.1 mmol/L 5,5-dithio-bis-(2-nitrobenzoic) acid, 0.25% Triton-X, 0.5 mmol/L oxaloacetate, 0.31 mmol/L acetyl coenzyme A, 5 µmol/L EDTA, 5 mmol/L triethanolamine hydrochloride, and 0.1 M Tris-HCl, pH 8.1. Subsequently, 20 µl of the mixed and homogenized content of the oxygraph chamber was added to 180 µl of the medium in a 96-well plate. The rate of change in the absorption of light (RAC) was measured spectrophotometrically at 412 nanometers and 30 degrees Celsius after 200 seconds. Data analysis and statistics The present study statistically tested the hypothesis that 3D-printed polymeric materials exert cytotoxic effects on human dermal fibroblasts (HDFs). Initially, the data were assessed for normality of distribution using the Shapiro-Wilk test. Subsequent to the evaluation of normality, statistical significance was determined through the implementation of the non-parametric Mann-Whitney U test. Statistical significance was attributed to differences when the p-value was less than 0.05. The results are presented as the ratio of the tested material to the untreated control group (CTRL), expressed in percentages, or as the mean ± standard deviation (SD). All statistical analyses were performed using OriginPro 2021 software (OriginLab Corporation, Northampton, Massachusetts, USA). RESULTS Cell morphology During the short exposure period (24 hours), no substantial alterations in cellular morphology or the presence of vacuoles, inclusions, or lipid droplets were observed when compared to the control group. During the long exposure period (7 days), a discernible change in morphology was observed in materials F7, F10, and P3 compared to the controls. In material F10 (FLEX), the presence of vacuoles, inclusions, or lipid droplets is clearly visible in the photograph (see detail in FIG. 3 ). In the other materials, no alterations in cellular morphology or cell growth patterns were observed during extended exposure periods (7 days), and the presence of vacuoles, inclusions, or lipid droplets was not detected in the cells. Cell proliferation A 24-hour exposure to all of the tested polymers did not result in any statistically significant changes in cell proliferation when compared with control cultures. The remaining PLA samples (F1–F4, P1) exhibited non-significant (n.s.) results, suggesting that short-term indirect contact did not disrupt proliferative activity. In a similar vein, the compounds CPE (F5), ABS (F6, F8, F9, S1), ASA (F7), PC-ABS (F11), PETG (P2), PC (P3), and PP (V1) exhibited no statistically significant deviations from the control values. FLEX (F10) demonstrated a marginal negative deviation, designated by a "–" symbol, though this deviation did not attain statistical significance. The collective findings indicate that short-term exposure to the examined 3D-printing polymers does not adversely affect cell proliferation under indirect contact conditions. In the context of prolonged exposure, spanning a duration of seven days, the majority of materials exhibited proliferation rates that were commensurate with those observed in control cultures. All samples of poly(lactic acid) (PLA) (F1–F4, P1), acrylonitrile butadiene styrene (ABS) (F6, F8, F9, S1), acrylonitrile (ASA) (F7), and poly(chlorotrifluoroethylene)-based poly(acrylonitrile) (PC-ABS) (F11) exhibited non-significant results, thereby confirming stable proliferative behavior over time. In contrast, several materials exhibited statistically significant increases in proliferation, including FLEX (F10, + 20.45%); p < 0.001), PETG (P2, + 23.73%); p < 0.001), PC (P3, + 11.75%); p < 0.001), and PP (V1, + 19.76%); p < 0.001). Furthermore, CPE (F5) exhibited a mild, non-significant increase, indicated by a "+" symbol. These findings suggest that while the majority of the tested polymers did not exert a detrimental effect on cell proliferation, prolonged exposure to certain materials, particularly FLEX, PETG, PC, and PP, was associated with an elevated proliferative response compared to the control group. Single cell fluorescence (CSF) In the context of a 24-hour exposure to the 3D-printing polymers under investigation, no statistically significant alterations in single-cell fluorescence were observed in comparison with the control cells. The classification of all PLA samples (F1–F4, P1) as non-significant (n.s.) confirmed the absence of acute cytotoxic effects. Conversely, CPE (F5) exhibited a marginal positive deviation, designated as "+", while ASA (F7) and FLEX (F10) demonstrated minor negative deviations, denoted as "–". Notably, these deviations lacked statistical significance. As indicated by the data, ABS samples (F6, F8, F9) and the PC-ABS blend (F11) exhibited minor, non-significant increases in fluorescence ("+"). In contrast, PETG (P2), PC (P3), and PP (V1) remained comparable to control levels. The short-term assay revealed no statistically significant suppression of fluorescence in any material. This finding indicates that brief indirect exposure to the tested polymers did not elicit detectable cytotoxic or metabolic effects in single-cell fluorescence measurements. In the course of extended observation periods (7 days), a quantifiable decline in single-cell fluorescence was observed among various polymers in comparison with the control cultures. Among the PLA samples, a significant reduction in fluorescence was observed in F1 (–10.35%; p < 0.001), F2 (–15.21%; p < 0.001), and F4 (–13.61%; p < 0.001). In contrast, F3 exhibited no change in fluorescence. Conversely, CPE (F5), ASA (F7), ABS (F6, F8, F9), PC-ABS (F11), and PC (P3) exhibited non-significant values. Conversely, a subset of materials exhibited a substantial decrease in fluorescence intensity. FLEX (F10, − 18.96%; p < 0.001), ABS (S1, − 21.04%; p < 0.001), PETG (P2, − 28.26%; p < 0.001) and PP (V1, − 27.42%; p < 0.001). These decreases are statistically significant relative to the control, indicating that long-term exposure to certain polymer types is associated with a substantial decline in single-cell fluorescence intensity, while the remaining materials retain values comparable to the control group. Cell viability In the context of a 24-hour exposure to the 3D-printing polymers under investigation, no statistically significant alterations in single-cell fluorescence were observed in comparison with the control cells. The classification of all PLA samples (F1–F4, P1) as non-significant (n.s.) confirmed the absence of acute cytotoxic effects. Conversely, CPE (F5) exhibited a marginal positive deviation, designated as "+", while ASA (F7) and FLEX (F10) demonstrated minor negative deviations, denoted as "–". Notably, these deviations lacked statistical significance. As indicated by the data, ABS samples (F6, F8, F9) and the PC-ABS blend (F11) exhibited minor, non-significant increases in fluorescence ("+"). In contrast, PETG (P2), PC (P3), and PP (V1) remained comparable to control levels. The short-term assay revealed no statistically significant suppression of fluorescence in any material. This finding indicates that brief indirect exposure to the tested polymers did not elicit detectable cytotoxic or metabolic effects in single-cell fluorescence measurements. In the course of extended observation periods (7 days), a quantifiable decline in single-cell fluorescence was observed among various polymers in comparison with the control cultures. Among the PLA samples, a significant reduction in fluorescence was observed in F1 (–10.35%; p < 0.001), F2 (–15.21%; p < 0.001), and F4 (–13.61%; p < 0.001). In contrast, F3 exhibited no change in fluorescence. Conversely, CPE (F5), ASA (F7), ABS (F6, F8, F9), PC-ABS (F11), and PC (P3) exhibited non-significant values. Conversely, a subset of materials exhibited a substantial decrease in fluorescence intensity. FLEX (F10, − 18.96%; p < 0.001), ABS (S1, − 21.04%; p < 0.001), PETG (P2, − 28.26%; p < 0.001) and PP (V1, − 27.42%; p < 0.001). These decreases are statistically significant relative to the control, indicating that long-term exposure to certain polymer types is associated with a substantial decline in single-cell fluorescence intensity, while the remaining materials retain values comparable to the control group. Table 2 – Table shows percentage changes in cell proliferation and metabolic activity after short-term (24 h) and long-term (7 days) exposure to different polymeric materials (values in %, relative to control). PLA and PET-G samples showed relatively minor effects, whereas some ABS derivatives (e.g., P2, P5) exhibited pronounced long-term reductions, particularly in metabolic activity and proliferation. Most polymers caused only minor short-term deviations, while PET-G (P2) showed more pronounced long-term reductions, suggesting potential alterations in membrane integrity or cell viability. Data also highlight that PLA and PET-G had only minor effects on proliferation, whereas some ABS derivatives (notably P2 and P5) caused marked reductions during prolonged exposure. PLA and ABS variants (P1, P3) showed only minor effects, whereas PET-G (P2) caused a pronounced long-term reduction in metabolic activity, indicating potential cytotoxic or stress-inducing effects. ID Material Cell Proliferation (SCF) Single cell fluorescence (SCPB) Metabolic Activity Short Δ (%) Long Δ (%) Short Δ (%) Long Δ (%) Short Δ (%) Long Δ (%) F1 PLA n.s. n.s. n.s. -10,35 + n.s. F2 PLA n.s. n.s. n.s. -15,21 + - F3 PLA n.s. n.s. n.s. - n.s. n.s. F4 PLA n.s. n.s. n.s. -13,61 n.s. - F5 CPE n.s. + + n.s. 10,99 n.s. F6 ABS n.s. n.s. n.s. n.s. 12,21 n.s. F7 ASA n.s. n.s. - n.s. n.s. + F8 ABS n.s. n.s. + n.s. 10,98 n.s. F9 ABS n.s. n.s. + n.s. + n.s. F10 FLEX - 20,45 - -18,96 n.s. -20,58 F11 PC-ABS n.s. n.s. + + 13,42 n.s. P1 PLA n.s. n.s. + n.s. 10,18 n.s. P2 PETG n.s. 23,73 - -28,26 10,60 -39,42 P3 PC n.s. 11,75 n.s. - 19,41 -15,27 S1 ABS n.s. n.s. n.s. -21,04 15,88 - V1 PP n.s. 19,76 n.s. -27,42 13,41 -21,30 HR Respirometry and Citrate synthase In order to verify the hypothesis that the observed changes in cell growth and metabolism are related to mitochondrial dysfunction (S. E. Lee et al., 2022 ), we measured mitochondrial respiration using HRR. A standardized SUIT protocol (see methods) and citrate synthase (CS) activity measurement were applied. The results, presented in Table 3 , have been normalized to the number of cells and compared to the control cells. Material F4 (PLA polymer with off-white pigmentation) exhibited a substantial decline in routine respiration (p < 0.001), while the other measured states merely indicated a tendency toward reduced respiration. Materials F5 (copolymer PET with off-white pigmentation) and F8 (ABS thermoplastic with off-white pigmentation) exhibited no significant impact on any of the measured respiratory states. Material F10 (i.e., modified thermoplastic polyurethane with a hardness of 98 Shore A and white pigmentation) exhibited the most pronounced changes, as indicated by a significant decrease in ROUT respiration and an increase in LEAK state, resulting in a reduction in ATP-coupled respiration (R-L; p < 0.001). Furthermore, the reserve capacity (E-R) of the cells exhibited a substantial augmentation. Materials P2 (glycol-modified PET polymer with black pigmentation) and P3 (polycarbonate polymer with off-white pigmentation) have been observed to stimulate cells to higher routine (p < 0.01) and R-L (p < 0.01) values. Table 3 Results High respirometry analysis (measured in millions of cells) as a response to long term (7 days) exposition to filament material. The number represents measured quantity ratio [%] of sample versus untreated control group. Bolt numbers represent statistically confirmed difference in sample compared to control group. n.s. represents non-significant difference, positive values indicate increased respiration value compared to untreated control cells, while negative values represent decreased activity. ROUT is a routine respiration, LEAK state corresponds shortcircuit of the proton cycle across the inner mt-membrane due to intrinsic uncoupling or dyscoupling, ETS is an electron transport system capacity, CIV is a Complex IV, R-L – ATP production, and E-R is cell respiration reserve. The values of respiration were found to be largely correlated with the values normalized to the number of cells, as indicated by the values normalized to citrate synthase (CS) activity (mitochondrial mass respiration; Table 4 ). A substantial increase in R-L state was observed for material F4 (p < 0.013). Materials F5 and F8 exhibited no significant impact on mitochondrial respiration, with the exception of an augmentation in reserve capacity observed in material F8 (p < 0.014). For the F10 model, a statistically significant decrease was observed in routine (p < 0.001), ETS (p < 0.001), CIV (p < 0.001), and R-L states (p < 0.001). Concurrently, an increase in reserve capacity (E-R; p < 0.03) was documented. Materials P2 and P3 exhibited no substantial disparities, with the exception of an augmentation in complex IV capacity, which was statistically significant for P2 (p < 0.005). Citrate synthase activity, normalized to cellular mass, exhibited a significant decrease in material F4, while it demonstrated an increase in F10 and P2. The remaining materials did not demonstrate alterations in mitochondrial quantity, as indicated by CS activity. ID Material ROUT LEAK ETS CIV R-L E-R F4 PLA -28,2 - - - - n.s. F5 CPE n.s. - n.s. + n.s. + F8 ABS n.s. n.s. n.s. n.s. n.s. n.s. F10 FLEX -143,2 29,9 + n.s. -727,7 34,9 P2 PETG 23,0 + + n.s. 24,8 + P3 PC 21,4 + + n.s. 23,8 n.s. Table 4 Results High respirometry analysis (expressed on mUI of CS enzyme) as a response to long term (7 days) exposition to filament material. The number represents measured quantity ratio [%] of sample versus untreated control group. Bolt numbers represent statistically confirmed difference in sample compared to control group. n.s. represents non-significant difference, positive values indicate increased respiration value compared to untreated control cells, while negative values represent decreased activity. ROUT is a routine respiration, LEAK state corresponds shortcircuit of the proton cycle across the inner mt-membrane due to intrinsic uncoupling or dyscoupling, ETS is an electron transport system capacity, CIV is a Complex IV, R-L – ATP production, and E-R is cell respiration reserve [mUI/Mbb] represents activity of citrate synthase per million cells. ID Material ROUT LEAK ETS CIV R-L E-R [mUI/Mbb] F4 PLA n.s. - + + 15,1 + -28,48 F5 CPE n.s. - n.s. + n.s. 15,4 n.s. F8 ABS n.s. n.s. n.s. n.s. n.s. n.s. n.s. F10 FLEX -222,1 n.s. -18,1 -28,9 -996,8 10,7 24,47 P2 PETG n.s. n.s. n.s. -12,9 n.s. n.s. 18,21 P3 PC n.s. n.s. n.s. - n.s. n.s. + Summary of observations Across all evaluated parameters—single-cell fluorescence, cell proliferation, metabolic activity, and mitochondrial respiration—the biological response to 3D-printing polymers was clearly material-specific and time-dependent. Short-term exposure did not induce measurable cytotoxic effects; however, prolonged exposure revealed distinct mitochondrial and metabolic impairments, particularly for FLEX (TPU), PETG, PC, and PP, which exhibited significant decreases in fluorescence and metabolic activity. HR respirometry confirmed these trends: FLEX (F10) elicited the most pronounced mitochondrial dysfunction, characterized by reduced routine respiration (ROUT) and ATP-coupled respiration (R–L) with a concomitant increase in LEAK and reserve capacity (E–R), consistent with energy uncoupling. PLA (F4) exhibited a substantial decrease in ROUT respiration, and following normalization to citrate synthase (CS) activity, a decline in mitochondrial efficiency was also observed, indicative of pigment-related metabolic suppression. The results of the study demonstrated that both PETG (P2) and PC (P3) exhibited increased rates of ROUT and R–L respiration, accompanied by elevated complex IV capacity. This finding suggests that the observed effects may be indicative of compensatory mitochondrial activation rather than overt toxicity. Conversely, CPE (F5), ABS (F8), and PC-ABS (F11) exhibited no substantial impact on respiratory states. However, citrate synthase activity analysis revealed a decrease in mitochondrial mass in F4 and an increase in F10 and P2. The collective results of these experiments demonstrate that prolonged indirect exposure to specific 3D-printing polymers—most notably FLEX, PETG, PC, and PP—induces measurable mitochondrial dysfunction and metabolic stress, whereas PLA, ABS, ASA, CPE, and PC-ABS maintain bioenergetic profiles that are nearly equivalent to the control group. DISCUSSION The present in vitro study examined the short-term (24 hours) and long-term (7 days) effects of commonly available polymers for 3D printing on human skin fibroblasts in indirect contact through a culture medium. While materials for additive manufacturing (3D printing) are frequently characterized as biocompatible (Burkhardt et al., 2022 ; Prakash et al., 2024 ; Zhu et al., 2025 ), our findings indicate that this assertion is only valid under certain constraints. The potential for materials to induce cellular toxicity can be observed not only in terms of viability and proliferation, but also in the processes intrinsic to cellular function, such as mitochondrial respiration. The findings of this study offer partial confirmation of existing knowledge and contribute to its extension. Of the materials that were tested, FLEX (F10), a modified thermoplastic polyurethane (TPU), demonstrated the highest level of cytotoxic activity. FLEX exerted a deleterious effect on cell morphology, as evidenced by the presence of vacuoles and lipid droplets. Proliferation and vitality were also adversely impacted. The observed discrepancy between the in vitro findings and the existing literature on the topic is noteworthy. Previous studies have indicated that FLEX (TPU) is a suitable suture material that does not cause inflammatory reactions (Haryńska et al., 2018 ; Vogels et al., 2017 ). Furthermore, a marked decline in mitochondrial routine respiration (ROUT) and elevated electron leakage (LEAK) was evident. This decline led to a substantial decrease in ATP phosphorylation capacity (R-L), a pivotal component of the cell's energy balance, which may potentially indicate a shift toward increased reliance on glycolytic pathways. These discrepancies can be attributed to the fact that although TPU is generally classified as a non-toxic and biocompatible polymer, its biological properties are highly dependent on its exact chemical composition. TPU optimized for 3D printing frequently contains additives such as pigments, UV stabilizers, flow modifiers, and additives that enhance the flexibility, plasticity, and processability of materials not present in the base granulate. The integration of lipophilic compounds into mitochondrial membranes or interaction with respiratory complexes has been demonstrated (Trnka, Elkalaf, and Anděl 2015 ). The adverse effects of various plasticizers on mitochondrial respiration have been well documented (Poitou et al. 2022 ). As postulated by Li et al. ( 2025 ), certain chemicals of a plastic-derived nature have been observed to elicit an increase in ROS. This phenomenon, in turn, has the potential to engender a diminution in electron flow and to foment the occurrence of leakage. Additionally, the printing process itself has the potential to be a source of toxicity due to the high temperatures of the print nozzle, which can lead to thermal degradation of the material and the release of harmful by-products. Of the materials tested, FLEX (F10), a modified thermoplastic polyurethane (TPU), was found to be the most cytotoxic. FLEX negatively affected cell morphology, including the presence of vacuoles and lipid droplets, as well as proliferation and vitality. This contrasts with literature data considering this material suitable for sutures that do not cause inflammatory reactions (Haryńska et al., 2018 ; Vogels et al., 2017 ). Additionally, we observed a significant decrease in mitochondrial respiratory capacity (ROUT) and increased electron leakage (LEAK). These changes result in an extreme decrease in ATP phosphorylation capacity (R-L), which is essential for maintaining the energy balance of the cell and may lead to a switch in metabolic processes. These discrepancies can be attributed to the fact that, although TPU is generally classified as nontoxic and biocompatible, its biological properties depend heavily on its exact chemical composition. TPU optimized for 3D printing often contains additives, such as pigments, UV stabilizers, and flow modifiers, as well as additives that increase flexibility, plasticity, and processability. These additives are not present in the base granulate. Lipophilic compounds can easily integrate into mitochondrial membranes or interact with respiratory complexes (Trnka et al., 2015 ). The adverse effects of various plasticizers on mitochondrial respiration are well-known (Poitou et al., 2022 ). Some plastic-derived chemicals increase ROS, which can decrease electron flow and promote leakage (Li et al., 2025 ). Furthermore, the printing process itself can be a source of cytotoxicity because the high temperatures of the print nozzle can cause the material to degrade thermally and release harmful byproducts. A body of research suggests that volatile organic compounds (VOCs) may be released during the process of thermoplastic polyurethane (TPU) printing. These compounds include tetrahydrofuran (THF), which has been identified as a potential carcinogen (Baguley et al., 2025 ; Byrley et al., 2020 ). Additionally, isocyanates, a class of chemicals involved in the printing process, have been shown to act as respiratory and skin sensitizers, potentially causing adverse health effects such as asthma, dermatitis, and irritation. Furthermore, isocyanates have been observed to be possible carcinogens, underscoring the need for further investigation into the safety of TPU printing processes. In polymers such as TPU, potential hazards emerge from residual unreacted isocyanates or their release during heating/processing (Grzęda, 2023 ; Pawlak et al., 2024 ; Restivo et al., 2024 ). Consequently, even 'clean' TPU can potentially exhibit significant toxic potential in a cellular environment if processed incorrectly, suggesting a hazard that warrants further toxicological assessment. Although Polylactic Acid (PLA) is generally considered a safe material (Hussain et al., 2024 ; Martino et al., 2009 ), a recent study suggests that it may not always be (Collin-Faure et al., 2024 ). Despite the absence of a discernible impact of the examined PLAs on the viability and proliferation of the utilized cell line, a substantial decline in routine mitochondrial respiration was observed in sample F4 (which exhibited a white pigment). This finding is in contrast with the results of another study, which reported an increase in oxidative phosphorylation in PLA (Maduka et al., 2023 ). This discrepancy may be attributable to variations in cell lines or the presence of specific additives in the tested filament. A notable positive finding is that the color pigments utilized exhibited no substantial impact on the evaluated cell properties. While Polyethylene terephthalate (PET) is regarded as a biocompatible material and has received FDA approval for certain medical applications (e.g., large blood vessels) (Çaykara et al., 2020 ; Gawlikowski et al., 2020 ), our data suggest that its chemically modified 3D printing analogue, PETG, may carry different toxicological properties. In the field of additive manufacturing, glycol-modified PET (PETG), a material that exhibits exceptional toughness and good thermal resistance, is employed. This material possesses versatile applications and is particularly well-suited for the fabrication of mechanical components. Although chemically modified PETG for 3D printing is regarded as being cytocompatible (Shilov et al., 2022 ), the results of this study demonstrated that it exerted a detrimental effect on the viability and proliferation of the tested skin fibroblasts. Furthermore, it exhibited increased basal respiration (ROUT) and capacity required for ATP phosphorylation from ADP. Conversely, chlorinated polyethylene (CPE), which is known for its high resistance to heat, chemicals, and UV radiation, demonstrated remarkable efficacy. The biocompatibility of the tested material, as measured by viability, proliferation, and mitochondrial respiration, was found to be practically comparable to the results of the unaffected control. According to the safety data sheets (SDS) of filament manufacturers, the use of CPE is safer than materials with similar chemical and UV resistance, such as ABS, due to its low VOCs emissions and ultrafine particles (UFPs). Given its observed high biocompatibility, CPE presents itself as a material of high interest for applications involving skin and food contact (BPA-free), though its full suitability requires further rigorous in vivo and regulatory assessment. A notable disadvantage of CPE is its substantial presence in micro- and nanoplastics pollution (Lotz et al., 2024 ; Ni et al., 2025 ; M. Wang et al., 2025 ). Acrylonitrile butadiene styrene (ABS) is widely regarded as biocompatible. However, the release of volatile organic compounds (VOCs) during the 3D printing process has been documented (Baguley et al., 2025 ; Chan et al., 2020 ; Wojnowski et al., 2022 ). Specifically, the presence of cytotoxic styrene and ethylbenzene has been observed on the surface of the final product. Consequently, we recommend that 3D-printed products be meticulously cleansed to remove residual VOCs, as demonstrated by the improved cellular tolerance observed in the ABS samples. One effective method for doing so is by subjecting the products to an ultrasonic cleaning process using pure ethanol, distilled water, or isopropyl alcohol (IPA). The results of this study indicate that the viability, proliferation, and mitochondrial respiration of used human lung fibroblasts were not significantly affected by the cleaning of ABS samples in this manner. This was observed in both short-term and long-term indirect contact scenarios when compared to control cells that remained unaffected. Polypropylene (PP) is also regarded as a biocompatible material (Hossain et al., 2024 ; Kelly et al., 2017 ; S. Lee et al., 2023 ). Short-term exposure led to a marginal increase in metabolic activity among exposed cells. Following extended exposure, cells exposed to PP demonstrated notable levels of material-induced cellular toxicity, a finding that aligns with prior observations (Burkhardt et al., 2022 ). As in other cases, this is likely attributable to the composition of additives such as UV stabilizers and flow modifiers. In a similar vein, the printing process has the potential to induce additional toxicity. Elevated print nozzle temperatures can result in thermal degradation of the material and the concomitant release of potentially harmful by-products. Polycarbonate (PC) is classified as a non-toxic plastic (Gómez-Gras et al., 2021 ; Weems et al., 2021 ). The findings of this study indicate that exposure to PC for a limited duration results in an augmentation of metabolic activity among the exposed cells. However, in the context of long-term exposure, a contrary response was observed, characterized by a decline in viability and proliferation. The observed toxicity of PC is consistent with the extant scientific knowledge regarding the release of bisphenol A (Cao et al., 2010b ; Coulier et al., 2010 ; Maragou et al., 2008 )). The release of additives from the polymer matrix is a complex process governed primarily by physical diffusion, in which chemically unbound substances migrate from the interior of the plastic to its surface and subsequently into the surrounding culture medium. The molecular weight and polarity of the additive influence the kinetics of this diffusion, with smaller molecules such as bisphenol A (BPA) migrating more easily (Cao et al., 2010a ; Maragou et al., 2008 ). Furthermore, the structural and crystalline characteristics of the polymer itself are foundational, as elevated material density impedes migration (Maddela et al., 2023 ). Research has demonstrated that external factors, including temperature and UV radiation, have the capacity to expedite the release of additives (Yaragatti & Patnaik, 2021 ). In the in vitro system utilized in this study, the release process is further influenced by media properties such as pH and the presence of organic substances (Yu et al., 2024 ). The presence of these released substances in the culture medium subsequently causes the observed toxic effects—such as the disruption of mitochondrial respiration and proliferation—which we documented. Therefore, it is essential to recognize that the assessment of plastic toxicity must be based not only on material identification but also on a comprehensive chemical profile of the substances released into the medium, as outlined in the "Study Limitations" section. The subsequent phase of our research will entail the testing of FLEX, PC, and PETG materials from various manufacturers. This will allow us to ascertain whether the tested material was an outlier or if this category of filament should be approached with a degree of circumspection. Furthermore, it is imperative to assess the impact of elevated temperatures during the 3D printing process on alterations in the chemical composition of the final material and the potential toxicity of the printed material (Yan et al., 2024 ). These alterations may not be evident in the original filament prior to the thermal process during 3D printing. STUDY LIMITATIONS Our study, while rigorous in its combination of the ISO 10993-5 standard with advanced mitochondrial analysis, carries inherent limitations that must be addressed, particularly regarding the translatability and chemical specificity of the findings. Firstly, a significant constraint, and the most critical limitation for translatability, is the reliance on a two-dimensional (2D) single-cell culture model utilizing primary human dermal fibroblasts. While HDFs are highly sensitive and relevant for initial screening of cellular toxicity, this configuration inherently lacks the complexity and physiological relevance of a three-dimensional (3D) tissue microenvironment. Furthermore, the study utilized an indirect interaction medium, which may not fully replicate direct skin contact with the printed object, including mechanical stimulation and temperature changes present in real-world wearable applications. The 2D model is incapable of fully replicating the complex cell-to-cell signaling, tissue structure, or diffusion characteristics of the skin. These characteristics may significantly modulate the ultimate in vivo toxicity of the polymer leachables. Secondly, the specific chemical identity and origin of the cytotoxic molecules remain ambiguous. The composition of commercial filament materials is frequently classified as proprietary information, thereby precluding definitive determination of the origin of observed toxicity, whether it stems from unknown cytotoxic additives, plasticizers, or dyes. The present study was unable to ascertain whether the observed toxicity is intrinsic to the source polymer or if it arises as a consequence of the cyclic heating and thermal degradation processes employed during 3D printing. Furthermore, the findings of this study are exclusive to the specific filament samples examined and may not be applicable to all products of the specified material from disparate manufacturers. Thirdly, although stringent measures were implemented to mitigate microbial contamination, the possibility of residual endotoxin presence—which could potentially act as a confounding factor—remains unassailable. The present study focused exclusively on the indirect interaction with dermal fibroblasts. A comprehensive toxicological assessment necessitates the evaluation of other key epidermal cells, including keratinocytes, melanocytes, and immune cells (Langerhans cells), to assess the full spectrum of potential biological responses and confirm the generalized risk to skin tissue. CONCLUSION The present study sought to investigate the influence of exposure of primary human dermal fibroblasts (HDF) to indirect contact with custom-designed 3D-printed inserts made from PLA, CPE, PETG, ABS, ASA, PC, PP, and FLEX on their viability, proliferation, metabolic activity, and mitochondrial respiration. The results strongly support the hypothesis regarding the cytotoxic potential of these materials, demonstrating a significant cytotoxic effect for FLEX (TPU), glycol-modified PET (PETG), and polycarbonate (PC) materials. For CPE, ABS, and PLA materials, the impact on viability and proliferation was negligible; however, even in these cases, slight changes in metabolic activity and mitochondrial respiration were observed. The findings suggest that materials that appear to be biocompatible may possess latent toxicity driven by leachable components. Consequently, our findings underscore the pressing need for more comprehensive toxicological evaluations and regulatory reassessments to ensure the safe utilization of these 3D printing polymers, particularly in biomedical and wearable device fabrication. These findings provide critical, foundational data for material selection aimed at minimizing cytotoxicity in the design of next-generation 3D-printed products. Abbreviations ABS Acrylonitrile butadiene styrene ADP Adenosine diphosphate AMA Antimycin A ASA Acrylonitrile Styrene Acrylate ATP Adenosine triphosphate AZD : CC Cell count CIV Complex IV CPE Thermoplastic chlorinated polyethylene elastomer CS Citrate synthase CTRL Control group DIFF Difference ratio DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic Acid EDTA Ethylenediaminetetraacetic acid ETS Electron Transport System ETSC Electron Transport System Capacity FCCP Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone FDA U.S. Food and Drug Administration FU Fluorescence units GRAS Generally Recognized as Safe HDF Human dermal fibroblast HRR High-resolution respirometry IPA Isopropyl alcohol IQ Interquartile ISO International Organization for Standardization IU International units IV Cytochrome c oxidase - Complex IV LCIM Live Cell Imaging Solution LEAK Mitochondrial Proton Leak MSDS Material Safety Data Sheets OMY Oligomycin PBS Phosphate-buffered saline PC Polycarbonate PEEK Polyether-ether-ketone PET Polyethylene terephthalate PETG Polyethylene terephthalate glycol PLA Polylactic acid PMMA Polymethyl methacrylate PP Polypropylene RAC Rate of absorbance change RH Relative humidity ROT Rotenone ROUT Routine mitochondria respiration ROX Residual oxygen consumption SCF Single cell fluorescence SCPB Single cell Prestoblue reduction rate SD Standard deviation SUIT Substrate-Uncoupler-Inhibitor-Titration TMPD N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride TPU Thermoplastic polyurethane UI International units USP United States Pharmacopeia VOC Volatile organic compounds Declarations CONFLICT OF INTEREST The authors have no competing interests to declare that are relevant to the content of this article. FUNDING DECLARATION This work has been funded by a grant from the Programme Johannes Amos Comenius under the Ministry of Education, Youth and Sports of the Czech Republic CZ.02.01.01/00/23_020/0008512. As set out in the Legal Act, beneficiaries must ensure that the open access to the published version or the final peer-reviewed manuscript accepted for publication is provided immediately after the date of publication via a trusted repository under the latest available version of the Creative Commons Attribution International Public Licence (CC BY) or a licence with equivalent rights. For long-text formats, CC BY-NC, CC BY-ND, CC BY-NC-ND or equivalent licenses could be applied. The work was also supported by the Cooperatio Program, Charles University and from European Regional Development Fund - Project „Fighting INfectious Diseases” (No. CZ.02.1.01/0.0/0.0/16_019/0000787). ACKNOWLEDGEMENTS We would like to thank prof. Jitka Kuncova, Ph.D. for providing access to mitochondrial laboratory facilities. We would also like to thank Prof. Zbyněk Tonar, Ph.D., for his valuable advice during the writing of this publication. DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS. Statement: In the course of preparing this work, the author(s) employed ChatGPT, Gemini, and DeepL for the purposes of language, grammar, and translation. Following the utilization of this service, the author conducted a thorough review and editing process to ensure the quality and integrity of the content. The author assumes full responsibility for the content of the published article. AUTHOR CONTRIBUTIONS J. D. (Jiří Dejmek): Conceived the study; secured funding; developed the 3D-printing protocol for inserts; performed filament chemical pre-analysis; provided the primary human dermal fibroblast cell line (HDF) and performed cell culture maintenance; designed and executed the cell viability and proliferation assays. J. D. also provided the intellectual supervision for the entire project, wrote the original draft of the manuscript, prepared all figures and tables, and critically reviewed and edited the final manuscript. J. J. (Jan Jedlička): Designed and performed the comprehensive mitochondrial respiration analysis; evaluated and interpreted the resulting bioenergetic data; contributed significantly to the Mitochondria Respiration sections of the manuscript; and participated in the discussion of the toxicological mechanisms. All authors read, discussed the results, and approved the final manuscript. Correspondence and requests for materials should be addressed to J.D. 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Environmental Pollution (Barking, Essex: 1987) , 343 , 123219. (2024). https://doi.org/10.1016/j.envpol.2023.123219 Zhu, Y. et al. 3D-Printed Polymeric Biomaterials for Health Applications. Adv. Healthc. Mater. 14 (1), e2402571. https://doi.org/10.1002/adhm.202402571 (2025). Zimmermann, L., Dierkes, G., Ternes, T. A., Völker, C. & Wagner, M. Benchmarking the in Vitro Toxicity and Chemical Composition of Plastic Consumer Products. Environmental Science Technology . 53 (19), 11467–11477. https://doi.org/10.1021/acs.est.9b02293 (2019). Additional Declarations No competing interests reported. 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16:38:29","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":208957,"visible":true,"origin":"","legend":"","description":"","filename":"ec77cadc8bb44ce39bdf7b5b984a5c4b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8095943/v1/94f353f6c5a04183777ed1c2.xml"},{"id":99286694,"identity":"f49f6ec9-f36c-4ba1-8d4f-8c8256c61c23","added_by":"auto","created_at":"2025-12-31 09:29:47","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":225980,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8095943/v1/3632a066897807d115c52fc0.html"},{"id":99320582,"identity":"9784a11d-f529-4ac0-bcd2-fd1e5d48cf4e","added_by":"auto","created_at":"2025-12-31 16:38:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":900309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic setup of the experiment. A 3D-printed insert made from the tested materials is placed into the culture well in such a way that it does not come into contact with the cultured cells. The material interacts with the cultured cell line indirectly through the culture medium. \u003cstrong\u003e(B)\u003c/strong\u003e Exact dimensions (in millimeters) of the 3D-printed insert. The dimensions and medium contact surface of the inserts are identical for each tested material. \u003cstrong\u003e(C)\u003c/strong\u003e Visualization of the placement of 3D-printed inserts from tested materials in culture wells. \u003cstrong\u003e(D)\u003c/strong\u003eExample of the setup from the in vitro experiment.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8095943/v1/67a6f968061cf07a09666338.jpeg"},{"id":99320430,"identity":"383aa2f8-8c8f-4ec3-bf4e-245a22f531d3","added_by":"auto","created_at":"2025-12-31 16:38:35","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1859049,"visible":true,"origin":"","legend":"\u003cp\u003ePhase-contrast microscopy images of human dermal fibroblasts after 7\u003csup\u003eth\u003c/sup\u003e day exposure to different polymeric materials. Untreated control cells (CTRL) display a typical elongated spindle-shaped morphology with uniform orientation and dense cell-to-cell contacts. Similar morphology and confluency are preserved after exposure to F4, F5, F8, P2, and P3. In contrast, cells cultured with F7 and especially F10 exhibit visible stress responses, including reduced cell density, altered orientation, and in the case of F10 also cytoplasmic vacuolization and irregular shapes. These findings indicate that while most tested materials are well tolerated, F7 and particularly F10 induce marked morphological changes consistent with cytotoxic effects. Scale bar: 200 μm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8095943/v1/6cf2ba1c7778e74364632822.jpeg"},{"id":99320474,"identity":"66c29699-1d8a-4556-8a5d-4e72ed377f58","added_by":"auto","created_at":"2025-12-31 16:38:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1083493,"visible":true,"origin":"","legend":"\u003cp\u003eDetail view of morphology changes in reaction on FLEX polymer. Untreated cells (CTRL, left) display a typical fibroblast morphology with elongated spindle-like shape, intact cytoplasm, and confluent growth pattern. In contrast, cells exposed to the tested material (F10, right) show marked morphological alterations, including cytoplasmic vacuolization in the perinuclear region, cellular swelling, and partial disruption of the elongated architecture, consistent with stress-induced or degenerative changes. Scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8095943/v1/cdd9a3a638892265a68ab477.jpeg"},{"id":99788628,"identity":"86031854-ad2d-4cbe-84af-c16f0a19915b","added_by":"auto","created_at":"2026-01-08 12:47:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5137284,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8095943/v1/471d5aca-b584-4f77-82f0-ec5a06d267a9.pdf"},{"id":99286682,"identity":"77e3f98f-8f0b-4b92-9d23-9eaf3b5bd25c","added_by":"auto","created_at":"2025-12-31 09:29:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1962724,"visible":true,"origin":"","legend":"","description":"","filename":"supplementumdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8095943/v1/905e9b7d78280604600bba4a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hidden cytotoxicity and mitochondrial dysfunction in 3D-printing polymers: evidence from FLEX, PETG and PC","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAdditive manufacturing, also known as 3D printing, has emerged as a prominent advanced technology that is fundamentally transforming the production of objects and structures across a wide range of applications, from rapid prototyping to the production of biomedical implants. Its application has proliferated across numerous sectors, encompassing domains such as healthcare, education, and sports. A particular and expanding category is that of wearable devices, which have evolved into a pivotal component of electronics (e.g., bracelets), textile technologies, and footwear design. A wide range of polymer materials in the form of filaments, such as PLA, PETG, CPE, ABS, and FLEX, are used for their production. These materials are available in a variety of colors and possess specific chemical properties that provide a high degree of flexibility in design and potential applications. A comprehensive study of recent advances in the additive manufacturing of smart textiles was conducted by (Manaia et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The research is centered on the domains of 3D and 4D printing technologies, with a focus on their potential for the development of future multifunctional textiles. (Song et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) presented a microfluidic diagnostic wearable device, created using 3D printing and integrating smart microfibers for medical purposes. This study highlights the potential of microfluidic systems for health diagnosis and monitoring.\u003c/p\u003e \u003cp\u003eGiven the expanding utilization of additive manufacturing for the fabrication of consumer goods and biomedical applications, it is imperative to evaluate the potential health risks stemming from the interaction of these polymer materials with biological systems. Specifically, the cytotoxic effects on human cells have not yet been sufficiently studied. A comprehensive evaluation of cellular toxicity is imperative to guarantee the safe and effective utilization of these materials, particularly in the context of direct or indirect contact with the human body (Derby, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Murphy \u0026amp; Atala, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tetsuka \u0026amp; Shin, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In their publication, (Tetsuka \u0026amp; Shin, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) critically reviewed the current trends in 3D-printed medical wearable devices and addressed potential health risks. Key concerns include the possibility of allergic reactions to printing materials and insufficient sterility of prints, which can lead to infection or implant rejection, compromising the safety and effectiveness of treatment (Augustine et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; He et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tappa \u0026amp; Jammalamadaka, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the realm of fundamental in vitro research, where 3D printing technology is employed to engineer specialized platforms for the study of cell motility, biological materials such as scaffolds, and microfluidic systems (Augustine et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Chander \u0026amp; Mahajan, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Farcas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Guttridge et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; J. M. Lee et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tuomi et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)knowledge of its potential cytotoxic effects is paramount.\u003c/p\u003e \u003cp\u003eThe scientific literature contains ample documentation of the toxicity of a range of polymer materials (Manoochehri et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zimmermann et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Notwithstanding this awareness, the dearth of crucial toxicological data in material safety data sheets (MSDSs) for additive manufacturing engenders valid concerns regarding their biological safety. Published data on the assessment of the material's cytotoxic properties for 3D printing applications are currently very limited, especially with regard to their filament form. Furthermore, filament manufacturers typically do not provide biocompatibility assessments in accordance with relevant standards, such as ISO 10993-5. These gaps in knowledge underscore the necessity for further systematic research. Research conducted by (Guttridge et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and (Satzer \u0026amp; Achleitner, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) has previously indicated that the utilization of prevalent materials and their surface chemical composition may exert an influence on cell viability, proliferation, and morphology. These findings underscore the pivotal function of material-cell interaction in ensuring the reliability and efficacy of cell experiments in biomedicine and bioengineering. The toxicity of color pigments has been demonstrated to pose a potential risk, thereby further complicating the safety assessment of finished products (Chia \u0026amp; Wu, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mota et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tetsuka \u0026amp; Shin, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA paucity of comprehensive information exists regarding the cytotoxic potential of materials commonly utilized in three-dimensional (3D) printing applications. In light of this deficit, the present study was conceived as a preliminary evaluative investigation of sixteen distinct polymers. The evaluation encompassed simulated scenarios of indirect contact, such as the utilization of 3D-printed wearable devices or footwear, or the employment of 3D-printed devices and inserts in the context of cell research. The following working hypothesis was formulated: the exposure of primary human dermal fibroblasts (HDF) to indirect contact with custom-designed 3D-printed inserts made from PLA, CPE, PETG, ABS, ASA, PC, PP, and FLEX will have a negative influence on their viability, proliferation, metabolic activity, and mitochondrial respiration (Gnaiger, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; S. E. Lee et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pr\u0026auml;bst et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Salin et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The degree of toxicity will depend on both the type of material and the duration of exposure (short-term, 24 hours; long-term, 7 days). HDF cells were selected as a robust mesenchymal model system that is highly relevant for evaluating human skin contact with plastics. The assessment of cellular toxicity was conducted in accordance with the established principles of ISO 10993-5:2009/A11:2025, employing an indirect contact method that ensured cells were not in direct physical contact with the polymer specimens. Instead, cells were continuously exposed to potential leachables that were diffusing from the material into the culture medium. In accordance with the ISO 10993-5 criteria, a reduction in cell viability of \u0026ge;\u0026thinsp;30% relative to the negative control was considered indicative of toxicity. The efficacy of indirect contact via inserts has been previously validated for a range of material types, including dental biomaterials (Babich et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), crosslinked polymers (M. O. Wang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and 3D-printed constructs (Rengarajan et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, it was deemed suitable for the present study to reproduce realistic scenarios of continuous leaching without direct material\u0026ndash;cell contact.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003e3D print filaments (materials)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the purpose of this study, a total of 16 different types of commercially available 3D printing filaments were selected to test the hypothesis. The selection criteria encompassed a range of polymer types, along with various color variants of these polymers. A comprehensive list of the tested filaments, accompanied by their respective manufacturers, is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Each sample was assigned a unique identifier in the first column of the table to facilitate reference and clarity in the presentation of the results. This structured approach ensures traceability and proper identification throughout the analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Table of polymer filaments used in this study. Material ID column serves for identification of material trough experiment and this publication. Column polymer express filament polymer material. Filament type express producer additional identification. Printed temperature in [\u0026deg;C] represent overall print temperature and heated temperature. Producer column represents filament producer..\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaterial ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolymer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFilament type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePrint temperature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eProducer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Crystal Clear\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e220/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Trafic White\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e220/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Trafic Black\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e220/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Natural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e220/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHG100 Extrafill Natural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e270/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Black\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e240/110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Natural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e260/110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Natural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e240/110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtrafill Transparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e240/110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFLEX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA98* Trafic White\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e240/50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePC-ABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNatural\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e270/110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFillamentum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJet Black\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e225/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePrusament\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePETG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePETG Jet Black\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e250/90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePrusament\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBlend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e275/115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePrusament\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMEDICAL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e240/100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSmartfill\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eV1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePP transparent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e240/80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eVerbatim\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTest sample preparation\u003c/h2\u003e \u003cp\u003eFor the in vitro testing, a custom-designed insert was fabricated. The dimensions of the specimen are presented in millimeters and illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. The insert consists of eight cylindrical hollow wells, precisely sized to fit into a single row of a 96-well plate. The height of the insert was designed to be less than the depth of the well to prevent direct contact between the tested polymeric material and the cellular components. The dimensions and medium contact surface of the inserts are identical for each material that was tested. The three-dimensional model of the insert was created using SolidWorks software (Dassault Syst\u0026egrave;mes, S.A.). The 3D model was subsequently prepared for printing using PrusaSlicer v2.4.1 software (Prusa3D, CZ). The inserts were fabricated using a standard, commercially available Prusa MK3s 3D printer (Prusa3D, CZ). In order to mitigate the risk of contamination from airborne particles, viruses, and bacteria, the printers were encased in a hermetically sealed enclosure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess bacterial sterility, ten samples were selected at random and placed into microbial cultivation broth after the completion of the 3D printing process. The samples were then incubated at 37\u0026deg;C, 100% relative humidity, and 5% carbon dioxide for a period of seven days. Subsequently, each sample was examined both macroscopically (for turbidity) and microscopically (for the presence of bacteria). No contamination was observed in any of the test samples. All the inserts were aseptically preserved without undergoing any additional sterilization until their utilization in the experiment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals\u003c/h3\u003e\n\u003cp\u003eIn the absence of an explicit statement to the contrary, all chemicals, assays, and cell culture plastics were procured from VWR International, a subsidiary of Avanton, located in Prague, Czech Republic.\u003c/p\u003e\n\u003ch3\u003eDevices\u003c/h3\u003e\n\u003cp\u003eThe immunofluorescence assessment of cells and their respective extracts was conducted using the Cytation5 plate reader (Agilent, USA) instrument with Gen5 software (version 3.10, Agilent, USA). Cell culture images were captured using an inverted microscope (Olympus IX75) with a Canon EOS 1300D camera and QuickPhoto PRO software (version 3.2 build 1887-II, Promedika, CZ). The measurement of mitochondrial respiration was performed on Oroboros O2k oxygraphs (Oroboros, Austria).\u003c/p\u003e\n\u003ch3\u003eCell culture and precultivation\u003c/h3\u003e\n\u003cp\u003eThe cell line of human dermal fibroblasts (HDF-ax3027) was obtained from Axol Bioscience (Easter Bush Hub, UK). HDF were cultivated in DMEM Medium (Biowest, L0101-500) with the following supplements: 10% (v/v) fetal bovine serum (Merck, F7524), 1% (v/v) penicillin-streptomycin (Merck, P4333), and 2.5 mmol/L L-glutamine (Merck, G7513). The cultivation took place at 37\u0026deg;C under 5% CO2 in a humidified incubator. For subculturing, cells were washed with Dulbecco phosphate-buffered saline (Merck, D8537) and then briefly incubated with TripLE Express (Gipco, 12605-028).\u003c/p\u003e\n\u003ch3\u003eExperiment protocol\u003c/h3\u003e\n\u003cp\u003eThe assessment of cellular toxicity was conducted in accordance with the established principles of ISO 10993-5:2009/A11:2025, employing an indirect contact method that ensured cells were not in direct physical contact with the polymer specimens. Instead, cells were continuously exposed to potential leachables that were diffusing from the material into the culture medium. To emulate acute exposure, cells were exposed to samples of the tested materials for a period of 24 hours. The simulation of chronic exposure was achieved by subjecting cells to samples of the tested materials for a period of seven days. The HDF cells were meticulously dispensed into 96-well plates (Avantor, model 734\u0026ndash;2327) with a density of 5 x 10\u0026sup3; cells per well. Throughout the experiment, the cells were cultivated under standard conditions in an incubator (air\u0026thinsp;+\u0026thinsp;5% CO₂). Subsequently, on the following day, 16 wells (N) were prepared for each material, and 3D-printed inserts were placed into these wells. The inserts were composed of the selected materials and were designed to be placed into wells containing planted cells, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(D). Each plate also contained a control group of unaffected cells (N\u0026thinsp;=\u0026thinsp;16). The culturing medium was changed on the fourth day of the experiment. For the HRR method (chronic exposure only), cells were cultured for seven days in a 250-ml bottle (Avantor, 734\u0026ndash;2809) in the same culture medium and under the same conditions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell morphology\u003c/h2\u003e \u003cp\u003eThe evaluation of cellular morphology was conducted through optical comparison, employing visual analysis of cellular structures under a microscope with appropriate magnification and illumination. In summary, images of cell culture were obtained on the second (acute) and seventh (chronic) days of the experiment. The subsequent morphological parameters between the control and affected cells will undergo comparison. The dimensions of cells have the potential to serve as indicators of alterations in the cytoskeleton, adhesion processes, or the cell cycle. The presence of vacuoles, inclusions, or lipid droplets in the cytoplasm of affected cells may be indicative of changes in metabolic activity or the presence of pathological changes. The degree of homogeneity in cell growth and the pattern of cell growth can provide information about proliferation, adhesion, and cell communication.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell proliferation\u003c/h3\u003e\n\u003cp\u003eTo assess and compare the growth of treated and control cells, live cell nucleus staining with NucBlue\u0026reg; (Invitrogen, Life Technologies, Prague, CZ) was utilized. On the second and seventh days of the experiment, filament sample inserts were extracted from the culture plates. Subsequently, the medium was aspirated, and the cells were rinsed with phosphate-buffered saline (PBS), which was also aspirated. Subsequently, 100 \u0026micro;l of the LCIM solution containing NucBlue at the manufacturer's recommended concentration (i.e., two drops per 1 ml of media) was added to the wells. The cells were then subjected to a culture process in an incubator maintained at a temperature of 37\u0026deg;C in an environment containing 5% CO2 for a duration of 20 minutes. Subsequently, the darkened samples were inserted into the Cytation5, and the number of nuclei in each well was precisely determined with a sample or control using the recommended protocol (λex\u0026thinsp;=\u0026thinsp;360 nm; λem\u0026thinsp;=\u0026thinsp;460 nm) of the Gen5 software.\u003c/p\u003e \u003cp\u003eSingle cell fluorescence (SCF) is defined as the average intensity of the fluorescence signal from a single cell. That is to say, it is the ability of NucBlue stain to penetrate the cell nucleus and intercalate into the DNA. It is also indicative of the presence of cell nucleus morphology abnormalities. The value of SCF was determined by the ratio of the absolute number of cells in a well to the total fluorescence response (FU) of a given well. The resultant value is designated as the SCF value.\u003c/p\u003e\n\u003ch3\u003eCell Viability\u003c/h3\u003e\n\u003cp\u003eTo compare the metabolic activity of the treated and control cells, the PrestoBlue\u0026reg; fluorescent reagent (Invitrogen, Life Technologies, Prague, CZ) was employed. PrestoBlue is a rapid cell viability indicator that utilizes the reducing power of live cells to convert resazurin to the fluorescent molecule, resorufin (O'Brien, Leiphrakpam, Xiao). The value of the absorption of the fluid above the precipitate, which represents the current concentration of blue resazurin and metabolized pink resorufin, was calculated according to the protocol established by the manufacturer, as detailed in the product documentation. The resulting value, denoted in the results as Single Cell PrestoBlue reduction rate (SCPB), represents the ratio of reduction capacity between the treated and control cells and is normalized per cell.\u003c/p\u003e \u003cp\u003eOn the second and seventh days, the inserts from the culture plates were removed. Subsequently, the medium was aspirated, and the cells were rinsed with phosphate-buffered saline (PBS). Subsequently, 100 \u0026micro;l of LCIM solution with PrestoBlue (at the manufacturer's recommended concentration, i.e., 100 \u0026micro;l per 1 ml of media) and NucBlue (at the manufacturer's recommended concentration, i.e., 2 drops per 1 ml of media) were added to the wells. The cells were then subjected to a culture process in an incubator maintained at a temperature of 37\u0026deg;C and a humidity level of 5% carbon dioxide for a duration of 120 minutes. Subsequently, the samples were inserted into the Cytation5, and using Gen 5 software, our protocol determined the absolute cell count in each well, the fluorescence value of the wells (FU), and the absorbance of wells with cells and wells without cells (blank). The data obtained was then utilized to calculate the results.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHigh Resolution respirometry HRR\u003c/h2\u003e \u003cp\u003eThe cells were then placed into four pre-calibrated oxygraphs (O2k, Oroboros, Austria), each containing two chambers with a volume of 2 ml at 37\u0026deg;C. Subsequent to the attainment of equilibrium, measurements were initiated using a standardized SUIT (Substrate-Uncoupler-Inhibitor-Titration) protocol. Subsequent to the ROUTINE state (respiration of unaffected intact cells), the following states were measured: LEAK: Respiratory compensation for a proton leak across the inner mitochondrial membrane following the inhibition of ATP synthase by oligomycin (OMY; 2.5 \u0026micro;mol/L, as determined by titration in a preliminary experiment). ETS: Electron Transport System Capacity, defined as maximal respiration subsequent to the uncoupling of oxidation and phosphorylation by FCCP (carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; 0.05 \u0026micro;mol/L, gradual titration). ROX: Residual oxygen consumption originating from sources other than the electron transport chain, titrated using rotenone (ROT; inhibitor of complex I; 0.5 \u0026micro;mol/L) and antimycin A (AMA; inhibitor of complex III; 2.5 \u0026micro;mol/L). The activity of complex IV was determined by titration with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride; an artificial substrate of complex IV; 0.5 mmol/l), ascorbate (a reducing agent for high auto-oxidation of TMPD; 2 mmol/l), and azide (AZD; an inhibitor of complex IV; 100 mmol/l). The data were processed using DatLab software, version 7.4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCitrate synthase activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe medium for determining citrate synthase activity consists of the following components: 0.1 mmol/L 5,5-dithio-bis-(2-nitrobenzoic) acid, 0.25% Triton-X, 0.5 mmol/L oxaloacetate, 0.31 mmol/L acetyl coenzyme A, 5 \u0026micro;mol/L EDTA, 5 mmol/L triethanolamine hydrochloride, and 0.1 M Tris-HCl, pH 8.1. Subsequently, 20 \u0026micro;l of the mixed and homogenized content of the oxygraph chamber was added to 180 \u0026micro;l of the medium in a 96-well plate. The rate of change in the absorption of light (RAC) was measured spectrophotometrically at 412 nanometers and 30 degrees Celsius after 200 seconds.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData analysis and statistics\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe present study statistically tested the hypothesis that 3D-printed polymeric materials exert cytotoxic effects on human dermal fibroblasts (HDFs). Initially, the data were assessed for normality of distribution using the Shapiro-Wilk test. Subsequent to the evaluation of normality, statistical significance was determined through the implementation of the non-parametric Mann-Whitney U test. Statistical significance was attributed to differences when the p-value was less than 0.05. The results are presented as the ratio of the tested material to the untreated control group (CTRL), expressed in percentages, or as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). All statistical analyses were performed using OriginPro 2021 software (OriginLab Corporation, Northampton, Massachusetts, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell morphology\u003c/h2\u003e \u003cp\u003eDuring the short exposure period (24 hours), no substantial alterations in cellular morphology or the presence of vacuoles, inclusions, or lipid droplets were observed when compared to the control group. During the long exposure period (7 days), a discernible change in morphology was observed in materials F7, F10, and P3 compared to the controls. In material F10 (FLEX), the presence of vacuoles, inclusions, or lipid droplets is clearly visible in the photograph (see detail in FIG. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the other materials, no alterations in cellular morphology or cell growth patterns were observed during extended exposure periods (7 days), and the presence of vacuoles, inclusions, or lipid droplets was not detected in the cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation\u003c/h2\u003e \u003cp\u003eA 24-hour exposure to all of the tested polymers did not result in any statistically significant changes in cell proliferation when compared with control cultures. The remaining PLA samples (F1\u0026ndash;F4, P1) exhibited non-significant (n.s.) results, suggesting that short-term indirect contact did not disrupt proliferative activity. In a similar vein, the compounds CPE (F5), ABS (F6, F8, F9, S1), ASA (F7), PC-ABS (F11), PETG (P2), PC (P3), and PP (V1) exhibited no statistically significant deviations from the control values. FLEX (F10) demonstrated a marginal negative deviation, designated by a \"\u0026ndash;\" symbol, though this deviation did not attain statistical significance. The collective findings indicate that short-term exposure to the examined 3D-printing polymers does not adversely affect cell proliferation under indirect contact conditions.\u003c/p\u003e \u003cp\u003eIn the context of prolonged exposure, spanning a duration of seven days, the majority of materials exhibited proliferation rates that were commensurate with those observed in control cultures. All samples of poly(lactic acid) (PLA) (F1\u0026ndash;F4, P1), acrylonitrile butadiene styrene (ABS) (F6, F8, F9, S1), acrylonitrile (ASA) (F7), and poly(chlorotrifluoroethylene)-based poly(acrylonitrile) (PC-ABS) (F11) exhibited non-significant results, thereby confirming stable proliferative behavior over time. In contrast, several materials exhibited statistically significant increases in proliferation, including FLEX (F10, +\u0026thinsp;20.45%); p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), PETG (P2, +\u0026thinsp;23.73%); p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), PC (P3, +\u0026thinsp;11.75%); p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and PP (V1, +\u0026thinsp;19.76%); p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, CPE (F5) exhibited a mild, non-significant increase, indicated by a \"+\" symbol. These findings suggest that while the majority of the tested polymers did not exert a detrimental effect on cell proliferation, prolonged exposure to certain materials, particularly FLEX, PETG, PC, and PP, was associated with an elevated proliferative response compared to the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSingle cell fluorescence (CSF)\u003c/h2\u003e \u003cp\u003eIn the context of a 24-hour exposure to the 3D-printing polymers under investigation, no statistically significant alterations in single-cell fluorescence were observed in comparison with the control cells. The classification of all PLA samples (F1\u0026ndash;F4, P1) as non-significant (n.s.) confirmed the absence of acute cytotoxic effects. Conversely, CPE (F5) exhibited a marginal positive deviation, designated as \"+\", while ASA (F7) and FLEX (F10) demonstrated minor negative deviations, denoted as \"\u0026ndash;\". Notably, these deviations lacked statistical significance. As indicated by the data, ABS samples (F6, F8, F9) and the PC-ABS blend (F11) exhibited minor, non-significant increases in fluorescence (\"+\"). In contrast, PETG (P2), PC (P3), and PP (V1) remained comparable to control levels. The short-term assay revealed no statistically significant suppression of fluorescence in any material. This finding indicates that brief indirect exposure to the tested polymers did not elicit detectable cytotoxic or metabolic effects in single-cell fluorescence measurements.\u003c/p\u003e \u003cp\u003eIn the course of extended observation periods (7 days), a quantifiable decline in single-cell fluorescence was observed among various polymers in comparison with the control cultures. Among the PLA samples, a significant reduction in fluorescence was observed in F1 (\u0026ndash;10.35%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), F2 (\u0026ndash;15.21%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and F4 (\u0026ndash;13.61%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, F3 exhibited no change in fluorescence. Conversely, CPE (F5), ASA (F7), ABS (F6, F8, F9), PC-ABS (F11), and PC (P3) exhibited non-significant values. Conversely, a subset of materials exhibited a substantial decrease in fluorescence intensity. FLEX (F10, \u0026minus;\u0026thinsp;18.96%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), ABS (S1, \u0026minus;\u0026thinsp;21.04%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), PETG (P2, \u0026minus;\u0026thinsp;28.26%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and PP (V1, \u0026minus;\u0026thinsp;27.42%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These decreases are statistically significant relative to the control, indicating that long-term exposure to certain polymer types is associated with a substantial decline in single-cell fluorescence intensity, while the remaining materials retain values comparable to the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCell viability\u003c/h2\u003e \u003cp\u003eIn the context of a 24-hour exposure to the 3D-printing polymers under investigation, no statistically significant alterations in single-cell fluorescence were observed in comparison with the control cells. The classification of all PLA samples (F1\u0026ndash;F4, P1) as non-significant (n.s.) confirmed the absence of acute cytotoxic effects. Conversely, CPE (F5) exhibited a marginal positive deviation, designated as \"+\", while ASA (F7) and FLEX (F10) demonstrated minor negative deviations, denoted as \"\u0026ndash;\". Notably, these deviations lacked statistical significance. As indicated by the data, ABS samples (F6, F8, F9) and the PC-ABS blend (F11) exhibited minor, non-significant increases in fluorescence (\"+\"). In contrast, PETG (P2), PC (P3), and PP (V1) remained comparable to control levels. The short-term assay revealed no statistically significant suppression of fluorescence in any material. This finding indicates that brief indirect exposure to the tested polymers did not elicit detectable cytotoxic or metabolic effects in single-cell fluorescence measurements.\u003c/p\u003e \u003cp\u003eIn the course of extended observation periods (7 days), a quantifiable decline in single-cell fluorescence was observed among various polymers in comparison with the control cultures. Among the PLA samples, a significant reduction in fluorescence was observed in F1 (\u0026ndash;10.35%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), F2 (\u0026ndash;15.21%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and F4 (\u0026ndash;13.61%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, F3 exhibited no change in fluorescence. Conversely, CPE (F5), ASA (F7), ABS (F6, F8, F9), PC-ABS (F11), and PC (P3) exhibited non-significant values. Conversely, a subset of materials exhibited a substantial decrease in fluorescence intensity. FLEX (F10, \u0026minus;\u0026thinsp;18.96%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), ABS (S1, \u0026minus;\u0026thinsp;21.04%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), PETG (P2, \u0026minus;\u0026thinsp;28.26%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and PP (V1, \u0026minus;\u0026thinsp;27.42%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These decreases are statistically significant relative to the control, indicating that long-term exposure to certain polymer types is associated with a substantial decline in single-cell fluorescence intensity, while the remaining materials retain values comparable to the control group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Table shows percentage changes in cell proliferation and metabolic activity after short-term (24 h) and long-term (7 days) exposure to different polymeric materials (values in %, relative to control). PLA and PET-G samples showed relatively minor effects, whereas some ABS derivatives (e.g., P2, P5) exhibited pronounced long-term reductions, particularly in metabolic activity and proliferation. Most polymers caused only minor short-term deviations, while PET-G (P2) showed more pronounced long-term reductions, suggesting potential alterations in membrane integrity or cell viability. Data also highlight that PLA and PET-G had only minor effects on proliferation, whereas some ABS derivatives (notably P2 and P5) caused marked reductions during prolonged exposure. PLA and ABS variants (P1, P3) showed only minor effects, whereas PET-G (P2) caused a pronounced long-term reduction in metabolic activity, indicating potential cytotoxic or stress-inducing effects.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaterial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCell Proliferation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e(SCF)\u003c/p\u003e \u003cp\u003eSingle cell fluorescence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e(SCPB)\u003c/p\u003e \u003cp\u003eMetabolic Activity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eShort Δ (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eLong Δ (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eShort Δ (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eLong Δ (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eShort Δ (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eLong Δ (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-10,35\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-15,21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-13,61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e10,99\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e12,21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e10,98\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFLEX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e20,45\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-18,96\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-20,58\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePC-ABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e13,42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e10,18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePETG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e23,73\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-28,26\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e10,60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-39,42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e11,75\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e19,41\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-15,27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-21,04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e15,88\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e19,76\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-27,42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e13,41\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-21,30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHR Respirometry and Citrate synthase\u003c/h2\u003e \u003cp\u003eIn order to verify the hypothesis that the observed changes in cell growth and metabolism are related to mitochondrial dysfunction (S. E. Lee et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we measured mitochondrial respiration using HRR. A standardized SUIT protocol (see methods) and citrate synthase (CS) activity measurement were applied.\u003c/p\u003e \u003cp\u003eThe results, presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, have been normalized to the number of cells and compared to the control cells. Material F4 (PLA polymer with off-white pigmentation) exhibited a substantial decline in routine respiration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the other measured states merely indicated a tendency toward reduced respiration. Materials F5 (copolymer PET with off-white pigmentation) and F8 (ABS thermoplastic with off-white pigmentation) exhibited no significant impact on any of the measured respiratory states. Material F10 (i.e., modified thermoplastic polyurethane with a hardness of 98 Shore A and white pigmentation) exhibited the most pronounced changes, as indicated by a significant decrease in ROUT respiration and an increase in LEAK state, resulting in a reduction in ATP-coupled respiration (R-L; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, the reserve capacity (E-R) of the cells exhibited a substantial augmentation. Materials P2 (glycol-modified PET polymer with black pigmentation) and P3 (polycarbonate polymer with off-white pigmentation) have been observed to stimulate cells to higher routine (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and R-L (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) values.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults High respirometry analysis (measured in millions of cells) as a response to long term (7 days) exposition to filament material. The number represents measured quantity ratio [%] of sample versus untreated control group. Bolt numbers represent statistically confirmed difference in sample compared to control group. n.s. represents non-significant difference, positive values indicate increased respiration value compared to untreated control cells, while negative values represent decreased activity. ROUT is a routine respiration, LEAK state corresponds shortcircuit of the proton cycle across the inner mt-membrane due to intrinsic uncoupling or dyscoupling, ETS is an electron transport system capacity, CIV is a Complex IV, R-L \u0026ndash; ATP production, and E-R is cell respiration reserve. The values of respiration were found to be largely correlated with the values normalized to the number of cells, as indicated by the values normalized to citrate synthase (CS) activity (mitochondrial mass respiration; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A substantial increase in R-L state was observed for material F4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.013). Materials F5 and F8 exhibited no significant impact on mitochondrial respiration, with the exception of an augmentation in reserve capacity observed in material F8 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.014). For the F10 model, a statistically significant decrease was observed in routine (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), ETS (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), CIV (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and R-L states (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Concurrently, an increase in reserve capacity (E-R; p\u0026thinsp;\u0026lt;\u0026thinsp;0.03) was documented. Materials P2 and P3 exhibited no substantial disparities, with the exception of an augmentation in complex IV capacity, which was statistically significant for P2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). Citrate synthase activity, normalized to cellular mass, exhibited a significant decrease in material F4, while it demonstrated an increase in F10 and P2. The remaining materials did not demonstrate alterations in mitochondrial quantity, as indicated by CS activity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaterial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eROUT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLEAK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCIV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR-L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eE-R\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePLA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-28,2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCPE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eABS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eFLEX\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-143,2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e29,9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-727,7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e34,9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePETG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e23,0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e24,8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e21,4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e23,8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults High respirometry analysis (expressed on mUI of CS enzyme) as a response to long term (7 days) exposition to filament material. The number represents measured quantity ratio [%] of sample versus untreated control group. Bolt numbers represent statistically confirmed difference in sample compared to control group. n.s. represents non-significant difference, positive values indicate increased respiration value compared to untreated control cells, while negative values represent decreased activity. ROUT is a routine respiration, LEAK state corresponds shortcircuit of the proton cycle across the inner mt-membrane due to intrinsic uncoupling or dyscoupling, ETS is an electron transport system capacity, CIV is a Complex IV, R-L \u0026ndash; ATP production, and E-R is cell respiration reserve [mUI/Mbb] represents activity of citrate synthase per million cells.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaterial\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eROUT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLEAK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCIV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eR-L\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eE-R\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e[mUI/Mbb]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePLA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e15,1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-28,48\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCPE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e15,4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eABS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eF10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eFLEX\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-222,1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-18,1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-28,9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-996,8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e10,7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e24,47\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePETG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-12,9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e18,21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eP3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ePC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSummary of observations\u003c/h2\u003e \u003cp\u003eAcross all evaluated parameters\u0026mdash;single-cell fluorescence, cell proliferation, metabolic activity, and mitochondrial respiration\u0026mdash;the biological response to 3D-printing polymers was clearly material-specific and time-dependent. Short-term exposure did not induce measurable cytotoxic effects; however, prolonged exposure revealed distinct mitochondrial and metabolic impairments, particularly for FLEX (TPU), PETG, PC, and PP, which exhibited significant decreases in fluorescence and metabolic activity. HR respirometry confirmed these trends: FLEX (F10) elicited the most pronounced mitochondrial dysfunction, characterized by reduced routine respiration (ROUT) and ATP-coupled respiration (R\u0026ndash;L) with a concomitant increase in LEAK and reserve capacity (E\u0026ndash;R), consistent with energy uncoupling. PLA (F4) exhibited a substantial decrease in ROUT respiration, and following normalization to citrate synthase (CS) activity, a decline in mitochondrial efficiency was also observed, indicative of pigment-related metabolic suppression. The results of the study demonstrated that both PETG (P2) and PC (P3) exhibited increased rates of ROUT and R\u0026ndash;L respiration, accompanied by elevated complex IV capacity. This finding suggests that the observed effects may be indicative of compensatory mitochondrial activation rather than overt toxicity. Conversely, CPE (F5), ABS (F8), and PC-ABS (F11) exhibited no substantial impact on respiratory states. However, citrate synthase activity analysis revealed a decrease in mitochondrial mass in F4 and an increase in F10 and P2. The collective results of these experiments demonstrate that prolonged indirect exposure to specific 3D-printing polymers\u0026mdash;most notably FLEX, PETG, PC, and PP\u0026mdash;induces measurable mitochondrial dysfunction and metabolic stress, whereas PLA, ABS, ASA, CPE, and PC-ABS maintain bioenergetic profiles that are nearly equivalent to the control group.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present in vitro study examined the short-term (24 hours) and long-term (7 days) effects of commonly available polymers for 3D printing on human skin fibroblasts in indirect contact through a culture medium. While materials for additive manufacturing (3D printing) are frequently characterized as biocompatible (Burkhardt et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Prakash et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), our findings indicate that this assertion is only valid under certain constraints. The potential for materials to induce cellular toxicity can be observed not only in terms of viability and proliferation, but also in the processes intrinsic to cellular function, such as mitochondrial respiration. The findings of this study offer partial confirmation of existing knowledge and contribute to its extension.\u003c/p\u003e \u003cp\u003eOf the materials that were tested, FLEX (F10), a modified thermoplastic polyurethane (TPU), demonstrated the highest level of cytotoxic activity. FLEX exerted a deleterious effect on cell morphology, as evidenced by the presence of vacuoles and lipid droplets. Proliferation and vitality were also adversely impacted. The observed discrepancy between the in vitro findings and the existing literature on the topic is noteworthy. Previous studies have indicated that FLEX (TPU) is a suitable suture material that does not cause inflammatory reactions (Haryńska et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vogels et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, a marked decline in mitochondrial routine respiration (ROUT) and elevated electron leakage (LEAK) was evident. This decline led to a substantial decrease in ATP phosphorylation capacity (R-L), a pivotal component of the cell's energy balance, which may potentially indicate a shift toward increased reliance on glycolytic pathways. These discrepancies can be attributed to the fact that although TPU is generally classified as a non-toxic and biocompatible polymer, its biological properties are highly dependent on its exact chemical composition. TPU optimized for 3D printing frequently contains additives such as pigments, UV stabilizers, flow modifiers, and additives that enhance the flexibility, plasticity, and processability of materials not present in the base granulate. The integration of lipophilic compounds into mitochondrial membranes or interaction with respiratory complexes has been demonstrated (Trnka, Elkalaf, and Anděl \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The adverse effects of various plasticizers on mitochondrial respiration have been well documented (Poitou et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As postulated by Li et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), certain chemicals of a plastic-derived nature have been observed to elicit an increase in ROS. This phenomenon, in turn, has the potential to engender a diminution in electron flow and to foment the occurrence of leakage. Additionally, the printing process itself has the potential to be a source of toxicity due to the high temperatures of the print nozzle, which can lead to thermal degradation of the material and the release of harmful by-products.\u003c/p\u003e \u003cp\u003eOf the materials tested, FLEX (F10), a modified thermoplastic polyurethane (TPU), was found to be the most cytotoxic. FLEX negatively affected cell morphology, including the presence of vacuoles and lipid droplets, as well as proliferation and vitality. This contrasts with literature data considering this material suitable for sutures that do not cause inflammatory reactions (Haryńska et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vogels et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, we observed a significant decrease in mitochondrial respiratory capacity (ROUT) and increased electron leakage (LEAK). These changes result in an extreme decrease in ATP phosphorylation capacity (R-L), which is essential for maintaining the energy balance of the cell and may lead to a switch in metabolic processes. These discrepancies can be attributed to the fact that, although TPU is generally classified as nontoxic and biocompatible, its biological properties depend heavily on its exact chemical composition. TPU optimized for 3D printing often contains additives, such as pigments, UV stabilizers, and flow modifiers, as well as additives that increase flexibility, plasticity, and processability. These additives are not present in the base granulate. Lipophilic compounds can easily integrate into mitochondrial membranes or interact with respiratory complexes (Trnka et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The adverse effects of various plasticizers on mitochondrial respiration are well-known (Poitou et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Some plastic-derived chemicals increase ROS, which can decrease electron flow and promote leakage (Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, the printing process itself can be a source of cytotoxicity because the high temperatures of the print nozzle can cause the material to degrade thermally and release harmful byproducts.\u003c/p\u003e \u003cp\u003eA body of research suggests that volatile organic compounds (VOCs) may be released during the process of thermoplastic polyurethane (TPU) printing. These compounds include tetrahydrofuran (THF), which has been identified as a potential carcinogen (Baguley et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Byrley et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, isocyanates, a class of chemicals involved in the printing process, have been shown to act as respiratory and skin sensitizers, potentially causing adverse health effects such as asthma, dermatitis, and irritation. Furthermore, isocyanates have been observed to be possible carcinogens, underscoring the need for further investigation into the safety of TPU printing processes. In polymers such as TPU, potential hazards emerge from residual unreacted isocyanates or their release during heating/processing (Grzęda, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pawlak et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Restivo et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, even 'clean' TPU can potentially exhibit significant toxic potential in a cellular environment if processed incorrectly, suggesting a hazard that warrants further toxicological assessment.\u003c/p\u003e \u003cp\u003eAlthough Polylactic Acid (PLA) is generally considered a safe material (Hussain et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Martino et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), a recent study suggests that it may not always be (Collin-Faure et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite the absence of a discernible impact of the examined PLAs on the viability and proliferation of the utilized cell line, a substantial decline in routine mitochondrial respiration was observed in sample F4 (which exhibited a white pigment). This finding is in contrast with the results of another study, which reported an increase in oxidative phosphorylation in PLA (Maduka et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This discrepancy may be attributable to variations in cell lines or the presence of specific additives in the tested filament. A notable positive finding is that the color pigments utilized exhibited no substantial impact on the evaluated cell properties.\u003c/p\u003e \u003cp\u003eWhile Polyethylene terephthalate (PET) is regarded as a biocompatible material and has received FDA approval for certain medical applications (e.g., large blood vessels) (\u0026Ccedil;aykara et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gawlikowski et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), our data suggest that its chemically modified 3D printing analogue, PETG, may carry different toxicological properties. In the field of additive manufacturing, glycol-modified PET (PETG), a material that exhibits exceptional toughness and good thermal resistance, is employed. This material possesses versatile applications and is particularly well-suited for the fabrication of mechanical components.\u003c/p\u003e \u003cp\u003eAlthough chemically modified PETG for 3D printing is regarded as being cytocompatible (Shilov et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the results of this study demonstrated that it exerted a detrimental effect on the viability and proliferation of the tested skin fibroblasts. Furthermore, it exhibited increased basal respiration (ROUT) and capacity required for ATP phosphorylation from ADP.\u003c/p\u003e \u003cp\u003eConversely, chlorinated polyethylene (CPE), which is known for its high resistance to heat, chemicals, and UV radiation, demonstrated remarkable efficacy. The biocompatibility of the tested material, as measured by viability, proliferation, and mitochondrial respiration, was found to be practically comparable to the results of the unaffected control. According to the safety data sheets (SDS) of filament manufacturers, the use of CPE is safer than materials with similar chemical and UV resistance, such as ABS, due to its low VOCs emissions and ultrafine particles (UFPs). Given its observed high biocompatibility, CPE presents itself as a material of high interest for applications involving skin and food contact (BPA-free), though its full suitability requires further rigorous in vivo and regulatory assessment. A notable disadvantage of CPE is its substantial presence in micro- and nanoplastics pollution (Lotz et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ni et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; M. Wang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAcrylonitrile butadiene styrene (ABS) is widely regarded as biocompatible. However, the release of volatile organic compounds (VOCs) during the 3D printing process has been documented (Baguley et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Chan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wojnowski et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Specifically, the presence of cytotoxic styrene and ethylbenzene has been observed on the surface of the final product. Consequently, we recommend that 3D-printed products be meticulously cleansed to remove residual VOCs, as demonstrated by the improved cellular tolerance observed in the ABS samples. One effective method for doing so is by subjecting the products to an ultrasonic cleaning process using pure ethanol, distilled water, or isopropyl alcohol (IPA). The results of this study indicate that the viability, proliferation, and mitochondrial respiration of used human lung fibroblasts were not significantly affected by the cleaning of ABS samples in this manner. This was observed in both short-term and long-term indirect contact scenarios when compared to control cells that remained unaffected.\u003c/p\u003e \u003cp\u003ePolypropylene (PP) is also regarded as a biocompatible material (Hossain et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kelly et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; S. Lee et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Short-term exposure led to a marginal increase in metabolic activity among exposed cells. Following extended exposure, cells exposed to PP demonstrated notable levels of material-induced cellular toxicity, a finding that aligns with prior observations (Burkhardt et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As in other cases, this is likely attributable to the composition of additives such as UV stabilizers and flow modifiers. In a similar vein, the printing process has the potential to induce additional toxicity. Elevated print nozzle temperatures can result in thermal degradation of the material and the concomitant release of potentially harmful by-products.\u003c/p\u003e \u003cp\u003ePolycarbonate (PC) is classified as a non-toxic plastic (G\u0026oacute;mez-Gras et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Weems et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The findings of this study indicate that exposure to PC for a limited duration results in an augmentation of metabolic activity among the exposed cells. However, in the context of long-term exposure, a contrary response was observed, characterized by a decline in viability and proliferation. The observed toxicity of PC is consistent with the extant scientific knowledge regarding the release of bisphenol A (Cao et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010b\u003c/span\u003e; Coulier et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Maragou et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003eThe release of additives from the polymer matrix is a complex process governed primarily by physical diffusion, in which chemically unbound substances migrate from the interior of the plastic to its surface and subsequently into the surrounding culture medium. The molecular weight and polarity of the additive influence the kinetics of this diffusion, with smaller molecules such as bisphenol A (BPA) migrating more easily (Cao et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e; Maragou et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Furthermore, the structural and crystalline characteristics of the polymer itself are foundational, as elevated material density impedes migration (Maddela et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Research has demonstrated that external factors, including temperature and UV radiation, have the capacity to expedite the release of additives (Yaragatti \u0026amp; Patnaik, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the in vitro system utilized in this study, the release process is further influenced by media properties such as pH and the presence of organic substances (Yu et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The presence of these released substances in the culture medium subsequently causes the observed toxic effects\u0026mdash;such as the disruption of mitochondrial respiration and proliferation\u0026mdash;which we documented. Therefore, it is essential to recognize that the assessment of plastic toxicity must be based not only on material identification but also on a comprehensive chemical profile of the substances released into the medium, as outlined in the \"Study Limitations\" section.\u003c/p\u003e \u003cp\u003eThe subsequent phase of our research will entail the testing of FLEX, PC, and PETG materials from various manufacturers. This will allow us to ascertain whether the tested material was an outlier or if this category of filament should be approached with a degree of circumspection. Furthermore, it is imperative to assess the impact of elevated temperatures during the 3D printing process on alterations in the chemical composition of the final material and the potential toxicity of the printed material (Yan et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These alterations may not be evident in the original filament prior to the thermal process during 3D printing.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSTUDY LIMITATIONS\u003c/h2\u003e \u003cp\u003eOur study, while rigorous in its combination of the ISO 10993-5 standard with advanced mitochondrial analysis, carries inherent limitations that must be addressed, particularly regarding the translatability and chemical specificity of the findings.\u003c/p\u003e \u003cp\u003eFirstly, a significant constraint, and the most critical limitation for translatability, is the reliance on a two-dimensional (2D) single-cell culture model utilizing primary human dermal fibroblasts. While HDFs are highly sensitive and relevant for initial screening of cellular toxicity, this configuration inherently lacks the complexity and physiological relevance of a three-dimensional (3D) tissue microenvironment. Furthermore, the study utilized an indirect interaction medium, which may not fully replicate direct skin contact with the printed object, including mechanical stimulation and temperature changes present in real-world wearable applications. The 2D model is incapable of fully replicating the complex cell-to-cell signaling, tissue structure, or diffusion characteristics of the skin. These characteristics may significantly modulate the ultimate in vivo toxicity of the polymer leachables.\u003c/p\u003e \u003cp\u003eSecondly, the specific chemical identity and origin of the cytotoxic molecules remain ambiguous. The composition of commercial filament materials is frequently classified as proprietary information, thereby precluding definitive determination of the origin of observed toxicity, whether it stems from unknown cytotoxic additives, plasticizers, or dyes. The present study was unable to ascertain whether the observed toxicity is intrinsic to the source polymer or if it arises as a consequence of the cyclic heating and thermal degradation processes employed during 3D printing. Furthermore, the findings of this study are exclusive to the specific filament samples examined and may not be applicable to all products of the specified material from disparate manufacturers.\u003c/p\u003e \u003cp\u003eThirdly, although stringent measures were implemented to mitigate microbial contamination, the possibility of residual endotoxin presence\u0026mdash;which could potentially act as a confounding factor\u0026mdash;remains unassailable.\u003c/p\u003e \u003cp\u003eThe present study focused exclusively on the indirect interaction with dermal fibroblasts. A comprehensive toxicological assessment necessitates the evaluation of other key epidermal cells, including keratinocytes, melanocytes, and immune cells (Langerhans cells), to assess the full spectrum of potential biological responses and confirm the generalized risk to skin tissue.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe present study sought to investigate the influence of exposure of primary human dermal fibroblasts (HDF) to indirect contact with custom-designed 3D-printed inserts made from PLA, CPE, PETG, ABS, ASA, PC, PP, and FLEX on their viability, proliferation, metabolic activity, and mitochondrial respiration. The results strongly support the hypothesis regarding the cytotoxic potential of these materials, demonstrating a significant cytotoxic effect for FLEX (TPU), glycol-modified PET (PETG), and polycarbonate (PC) materials. For CPE, ABS, and PLA materials, the impact on viability and proliferation was negligible; however, even in these cases, slight changes in metabolic activity and mitochondrial respiration were observed. The findings suggest that materials that appear to be biocompatible may possess latent toxicity driven by leachable components. Consequently, our findings underscore the pressing need for more comprehensive toxicological evaluations and regulatory reassessments to ensure the safe utilization of these 3D printing polymers, particularly in biomedical and wearable device fabrication. These findings provide critical, foundational data for material selection aimed at minimizing cytotoxicity in the design of next-generation 3D-printed products.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eABS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcrylonitrile butadiene styrene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eADP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenosine diphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAMA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimycin A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eASA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcrylonitrile Styrene Acrylate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eATP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenosine triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAZD\u003c/b\u003e:\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCell count\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCIV\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComplex IV\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCPE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThermoplastic chlorinated polyethylene elastomer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCitrate synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCTRL\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDIFF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifference ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's Modified Eagle Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic Acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEDTA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEthylenediaminetetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eETS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eElectron Transport System\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eETSC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eElectron Transport System Capacity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFCCP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbonyl cyanide p-trifluoro-methoxyphenyl hydrazone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFDA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eU.S. Food and Drug Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFluorescence units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGRAS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGenerally Recognized as Safe\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHDF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman dermal fibroblast\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHRR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-resolution respirometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIPA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIsopropyl alcohol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIQ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterquartile\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eISO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInternational Organization for Standardization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInternational units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIV\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochrome c oxidase - Complex IV\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLCIM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLive Cell Imaging Solution\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLEAK\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial Proton Leak\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMSDS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMaterial Safety Data Sheets\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOMY\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOligomycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolycarbonate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePEEK\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyether-ether-ketone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePET\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyethylene terephthalate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePETG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyethylene terephthalate glycol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePLA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolylactic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePMMA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymethyl methacrylate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolypropylene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRAC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRate of absorbance change\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRelative humidity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eROT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRotenone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eROUT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoutine mitochondria respiration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eROX\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eResidual oxygen consumption\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSCF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle cell fluorescence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSCPB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle cell Prestoblue reduction rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSUIT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSubstrate-Uncoupler-Inhibitor-Titration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTMPD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTPU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThermoplastic polyurethane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInternational units\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eUSP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUnited States Pharmacopeia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eVOC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVolatile organic compounds\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eCONFLICT OF INTEREST\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003eFUNDING DECLARATION\u003c/p\u003e\n\u003cp\u003eThis work has been funded by a grant from the Programme Johannes Amos Comenius under the Ministry of Education, Youth and Sports of the Czech Republic CZ.02.01.01/00/23_020/0008512. As set out in the Legal Act, beneficiaries must ensure that the open access to the published version or the final peer-reviewed manuscript accepted for publication is provided immediately after the date of publication via a trusted repository under the latest available version of the Creative Commons Attribution International Public Licence (CC BY) or a licence with equivalent rights. For long-text formats, CC BY-NC, CC BY-ND, CC BY-NC-ND or equivalent licenses could be applied. The work was also supported by the Cooperatio Program, Charles University and from European Regional Development Fund - Project \u0026bdquo;Fighting INfectious Diseases\u0026rdquo; (No. CZ.02.1.01/0.0/0.0/16_019/0000787).\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGEMENTS\u003c/p\u003e\n\u003cp\u003eWe would like to thank prof. Jitka Kuncova, Ph.D. for providing access to mitochondrial laboratory facilities. We would also like to thank Prof. Zbyněk Tonar, Ph.D., for his valuable advice during the writing of this publication.\u003c/p\u003e\n\u003cp\u003eDECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS.\u003c/p\u003e\n\u003cp\u003eStatement: In the course of preparing this work, the author(s) employed ChatGPT, Gemini, and DeepL for the purposes of language, grammar, and translation. Following the utilization of this service, the author conducted a thorough review and editing process to ensure the quality and integrity of the content. The author assumes full responsibility for the content of the published article.\u003c/p\u003e\n\u003cp\u003eAUTHOR CONTRIBUTIONS\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. D. (Jiř\u0026iacute; Dejmek):\u0026nbsp;\u003c/strong\u003eConceived the study; secured funding; developed the 3D-printing protocol for inserts; performed filament chemical pre-analysis; provided the primary human dermal fibroblast cell line (HDF) and performed cell culture maintenance; designed and executed the cell viability and proliferation assays. J. D. also provided the intellectual supervision for the entire project, wrote the original draft of the manuscript, prepared all figures and tables, and critically reviewed and edited the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. J. (Jan Jedlička):\u0026nbsp;\u003c/strong\u003eDesigned and performed the comprehensive mitochondrial respiration analysis; evaluated and interpreted the resulting bioenergetic data; contributed significantly to the Mitochondria Respiration sections of the manuscript; and participated in the discussion of the toxicological mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAll authors\u003c/strong\u003e read, discussed the results, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to J.D.\u003c/p\u003e\n\u003cp\u003eORCID\u003c/p\u003e\n\u003cp\u003eJiř\u0026iacute; Dejmek 0000-0003-0542-5238\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJan Jedlička 0000-0001-5999-6875\u003c/p\u003e\n\u003cp\u003eDATA AVAILABILITY\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAugustine, E. K. et al. Resorbable 3D-Printed Osteosynthetic Plates for Rib Fracture Repair. \u003cem\u003eAdv. Healthc. Mater.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (16), e2500409. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adhm.202500409\u003c/span\u003e\u003cspan address=\"10.1002/adhm.202500409\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabich, H., Liebling, E. J., Burger, R. F., Zuckerbraun, H. L. \u0026amp; Schuck, A. G. Choice of DMEM, formulated with or without pyruvate, plays an important role in assessing the in vitro cytotoxicity of oxidants and prooxidant nutraceuticals. \u003cem\u003eIn Vitro Cellular\u003c/em\u003e \u0026amp; \u003cem\u003eDevelopmental Biology\u003c/em\u003e - \u003cem\u003eAnimal\u003c/em\u003e, \u003cem\u003e45\u003c/em\u003e(5), 226\u0026ndash;233. (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11626-008-9168-z\u003c/span\u003e\u003cspan address=\"10.1007/s11626-008-9168-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaguley, D. A., Evans, G. S., Bard, D., Monks, P. S. \u0026amp; Cordell, R. L. Review of volatile organic compound (VOC) emissions from desktop 3D printers and associated health implications. \u003cem\u003eJournal Exposure Science Environmental Epidemiology\u003c/em\u003e. 1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41370-025-00778-y\u003c/span\u003e\u003cspan address=\"10.1038/s41370-025-00778-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurkhardt, F. et al. 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Benchmarking the in Vitro Toxicity and Chemical Composition of Plastic Consumer Products. \u003cem\u003eEnvironmental Science Technology\u003c/em\u003e. \u003cb\u003e53\u003c/b\u003e (19), 11467\u0026ndash;11477. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.est.9b02293\u003c/span\u003e\u003cspan address=\"10.1021/acs.est.9b02293\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"3D print, cytotoxicity, Filament, PLA, PET, ABS, ASA, PC, PP, TPU, CPE, FLEX, fibroblast, cell viability, mitochondria, respirometry","lastPublishedDoi":"10.21203/rs.3.rs-8095943/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8095943/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdditive manufacturing, also known as 3D printing, is a rapidly evolving technology that is profoundly impacting consumer products and biomedical applications. The persistent lack of essential toxicological data in material safety data sheets (MSDS) for additive manufacturing raises legitimate concerns regarding the biological safety of the polymers utilized in 3D printing. In this study, the cytotoxic potential of eight widely available filaments\u0026mdash;polylactic acid (PLA), polyethylene terephthalate (PETG), chlorinated polyethylene (CPE), polycarbonate (PC), acrylonitrile butadiene styrene (ABS), polypropylene (PP), and flexible polyurethane (FLEX)\u0026mdash;was examined using an ISO 10993-5 compliant indirect contact assay on primary human dermal fibroblasts. Cells were exposed to leachables diffusing from 3D-printed inserts for 24 hours or 7 days, and viability, proliferation, metabolic activity, and mitochondrial respiration were assessed. The investigation revealed that FLEX (thermoplastic polyurethane), PETG, and PC induced significant cytotoxic effects, including impaired proliferation, altered morphology, and disrupted mitochondrial respiration. Conversely, PLA, ABS, and CPE demonstrated minimal impact under the tested conditions. The observed toxicity is likely associated with additives, pigments, and plasticizers, such as isocyanates or volatile organic compounds (VOCs). These compounds are released during the thermal degradation of the material during printing. Specifically, the toxicity profile aligns with the known hazards of residual isocyanates in FLEX, glycol modifications in PETG, and the known release of bisphenol A and related compounds from PC. These findings suggest that materials commonly regarded as biocompatible may exhibit hidden toxicity due to additives or degradation by-products generated during the printing process. The findings of this study underscore the imperative for a systematic toxicological evaluation and stringent regulatory oversight of 3D-printing polymers, particularly given their pervasive use in consumer contact applications\u0026mdash;including wearables (such as customized shoes and wristbands) and items intended for vulnerable populations (such as infant and toddler toys)\u0026mdash;where direct and long-term exposure indicates a potential, yet unrecognized, risk to public health.\u003c/p\u003e","manuscriptTitle":"Hidden cytotoxicity and mitochondrial dysfunction in 3D-printing polymers: evidence from FLEX, PETG and PC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 09:29:42","doi":"10.21203/rs.3.rs-8095943/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-25T11:16:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-07T14:19:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293961003037226298584680963340617710694","date":"2025-11-30T17:54:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271080702343817083759953443479383587311","date":"2025-11-29T07:44:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-26T06:46:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-26T06:45:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-19T11:26:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-17T09:20:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-17T09:16:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c34aab5a-b7eb-476b-9ec9-ea5472a5ccfb","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":59227476,"name":"Physical sciences/Chemistry"},{"id":59227477,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2026-05-14T09:40:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-31 09:29:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8095943","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8095943","identity":"rs-8095943","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}