Abstract
Three-dimensional (3D) printing and bioprinting technologies are increasingly used in tissue engineering and in the development of personalized medical devices with tailored biological and mechanical properties. Despite their advantages, these technologies may involve exposure to chemical compounds with uncertain biological effects, including potential endocrine-disrupting activity. This study aims to evaluate the hormonal activity and cytotoxicity of various materials commonly used for 3D printing technologies, including thermoplastics and synthetic materials (PLA, ABS, PCL, b-TPUe, PVA, PEGDA), and biomaterials (collagen, alginate, hyaluronic acid, agarose, silk fibroin, GelMA, HAMA). Hormonal activity was evaluated using the E-screen assay for estrogenic and anti-estrogenic activity, and the PALM assay for androgenic and anti-androgenic activity. Cytotoxic effects are evaluated by assessing cellular metabolic viability using the MTT assay. No detectable estrogenic, anti-estrogenic, androgenic, or anti-androgenic activity, nor any signs of cytotoxicity, were observed within the experimental conditions analysed. This study represents the first analysis of some specific endocrine-related endpoints of a wide range of 3D printing and bioprinting materials, and should not be interpreted as definitive proof of the complete absence of endocrine activity. Further studies addressing additional toxicological parameters and long-term assessments are required to comprehensively characterize their safety and suitability.
Keywords: hormonal activity, endocrine disruption, E-screen assay, 3D printing, bioprinting, biomaterials
1. Introduction
Three-dimensional (3D) printing and bioprinting are transforming current clinical practice by enabling tissue fabrication and the generation of 3D structures with biological and mechanical properties suitable for restoring tissue and organ function. In addition, these technologies facilitate the design of personalized medical devices generated from the patient’s own cells, allowing the creation of personalized functional constructs [1]. 3D printing enables the layer-by-layer fabrication of structures, while bioprinting applies this approach to bioinks formulated with natural and/or synthetic materials that can encapsulate cells, enabling the generation of constructs with tailored biological and mechanical properties [2,3].
The use of these emerging technologies may involve exposure to chemical compounds with potentially uncharacterized biological effects. For example, thin polycarbonate splints, which are characterized by high strength, hardness, and durability, may release relevant amounts of bisphenol A (BPA), the main component of the polymer and a well-known endocrine disruptor [4,5]. Exposure to BPA and other endocrine-disrupting chemicals from medical devices has been previously reported [4,6].
The presence of endocrine disruptors in these new materials may therefore pose potential health risks, especially if exposure occurs during critical developmental stages or is prolonged over time. Such exposure may contribute to diseases linked to disruptions in hormonal homeostasis [7,8]. To address this concern, we evaluated the hormonal activity and cytotoxicity of thirteen natural and synthetic materials commonly employed in the generation of bioprinted tissues, organs and medical devices [3].
2. Materials and Methods
The materials analyzed included polylactic acid (PLA) [9,10], acrylonitrile butadiene styrene (ABS) [10], polycaprolactone (PCL) [11], 1,4-butanediol thermoplastic polyurethane elastomer (b-TPUe) [12,13], polyvinyl alcohol (PVA) [14], poly(ethylene glycol) diacrylate (PEGDA) [15], collagen [16,17], alginate [14,16,18], hyaluronic acid [19], agarose [17], silk fibroin [18], methacrylated gelatine (GelMA) [15,20] and methacrylated hyaluronic acid (HAMA) [20,21]. Briefly, the tested materials were dissolved according to their solubility properties. PLA, ABS, PCL, and b-TPUe were dissolved in dimethyl sulfoxide (DMSO); PEGDA, collagen, alginate, hyaluronic acid, agarose, silk fibroin, GelMA, and HAMA were dissolved in phosphate-buffered saline (PBS); and PVA was dissolved in double-distilled water (DDW). When required, specific dissolution conditions were applied (e.g., incubation at 4 °C, 37 °C or 60 °C, heating, and/or sonication).
Hormonal activities were evaluated through the E-screen and PALM assays as described by Molina-Molina et al. [22,23]. Briefly, estrogenic and anti-estrogenic activities (E-Screen) were assessed in the human breast cancer cell line MCF-7, a well-established estrogen-responsive model, by measuring cell proliferation in the presence or absence of 17β-estradiol. Anti-estrogenic activity was determined by the ability of the compounds to inhibit estradiol-induced proliferation. Androgenic and anti-androgenic activities were evaluated using the androgen-dependent PALM cells by measuring changes in luciferase activity following exposure to the test compounds. Anti-androgenic effects were determined by coincubation with the reference androgen metiltrienolone (R1881). Additionally, cellular metabolic viability following exposure to the tested compounds was assessed using the MTT assay, based on the modified method of Denizot and Lang [24]. Stock solutions of the tested materials were subsequently serially diluted in the corresponding test culture medium before biological testing. For all experimental conditions, including the test materials and positive controls, the final solvent concentration in the culture medium did not exceed 0.1% (v/v). Moreover, each treatment was compared with its corresponding vehicle control, ensuring that any observed biological effects were attributable to the tested materials rather than to the solvent. Maximum concentrations of the tested materials were within or slightly below those reported for bioinks in the literature [14,15,16,17,18,19,20,21], while thermoplastic polymers were tested at a molar concentration equal to the maximum used for the biopolymers. This approach enabled testing under relevant conditions for bioprinting applications while maintaining fully dissolved solutions.
All experiments were performed in three independent experiments, with triplicate (E-screen and MTT) or quadruplicate (PALM) measurements for each concentration. Data are presented as mean ± standard deviation (SD). Statistical significance was assessed using one-way ANOVA followed by Dunnett’s post hoc test, with p < 0.05 considered statistically significant.
3. Results and Discussion
The materials analyzed included synthetic polymers: PLA [9,10], ABS [10], PCL [11], b-TPUe [12,13], PVA [14] and PEGDA [15], as well as natural biomaterials, including chemically modified biomaterials commonly used in bioinks: collagen [16,17], alginate [14,16,18], hyaluronic acid [19], agarose [17], silk fibroin [18], GelMA [15,20] and HAMA [20,21].
The effects of the evaluated materials in the E-screen assay are summarized in Table 1. Tested concentrations were selected according to the solubility of each material and reported concentrations for bioinks in the literature [14,15,16,17,18,19,20,21]. Across all tested concentrations, none of the materials showed detectable estrogenic activity comparable to that of the positive control, with values remaining close to the baseline, even at the higher concentrations tested. When the materials were grouped by type, no clear trend towards increased activity at higher concentrations was observed when synthetic materials were compared with natural ones. For anti-estrogenic activity, all materials yielded values comparable to the positive control, with no concentration-dependent decrease in proliferation observed, and no material types showing a tendency toward anti-estrogenic behaviour. The results in the PALM assay showed similar results (Table 2), with all tested materials yielding androgenic activity values close to the negative controls. When the compounds were tested for their ability to antagonize human androgen receptor (hAR) in our cell model, no antagonistic activity was detected. None of the tested materials produced a meaningful dose-dependent change in activity, nor was any clear difference observed between synthetic and natural materials. All positive and negative controls yielded the expected results, confirming the validity of the assays.
Table 1.
Proliferative and anti-proliferative effects of 13 bioprinting materials measured by the E-screen assay in MCF-7 cells.
| PLA | ABS | PCL | b-TPUe | PEDGA | Hyaluronic Acid | Agarose | Silk Fibroin | GelMa | HAMA | PVA | Collagen | Alginate | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Estrogenic activity | C+ | 6.36 * ± 0.06 | 6.46 * ± 0.34 | 6.45 * ± 0.22 | 6.53 * ± 0.21 | 6.36 * ± 0.17 | 6.51 * ± 0.04 | 6.39 * ± 0.36 | 6.53 * ± 0.16 | 6.24 * ± 0.11 | 6.17 * ± 0.11 | 6.51 * ± 0.04 | 6.76 * ± 0.10 | 6.51 * ± 0.21 |
| C− | 1.00 ± 0.04 | 1.00 ± 0.11 | 1.00 ± 0.06 | 1.00 ± 0.03 | 1.00 ± 0.04 | 1.00 ± 0.01 | 1.00 ± 0.01 | 1.00 ± 0.01 | 1.00 ± 0.02 | 1.00 ± 0.04 | 1.00 ± 0.03 | 1.00 ± 0.10 | 1.00 ± 0.01 | |
| 10−9 M | N.T. | 1.04 ± 0.02 | 1.03 ± 0.03 | 1.09 ± 0.05 | ||||||||||
| 3 × 10−9 M | N.T. | 1.10 ± 0.03 | 1.09 ± 0.03 | 1.11 ± 0.06 | ||||||||||
| 10−8 M | 1.02 ± 0.03 | 1.05 ± 0.05 | 1.03 ± 0.04 | 1.04 ± 0.01 | 1.08 ± 0.01 | 1.02 ± 0.02 | 1.06 ± 0.01 | 1.04 ± 0.02 | 1.06 ± 0.01 | 1.01 ± 0.01 | 1.07 ± 0.03 | 1.13 ± 0.04 | 1.11 ± 0.01 | |
| 3 × 10−8 M | 1.04 ± 0.03 | 1.05 ± 0.06 | 1.04 ± 0.04 | 1.09 ± 0.01 | 1.09 ± 0.02 | 1.07 ± 0.02 | 1.07 ± 0.01 | 1.06 ± 0.03 | 1.12 ± 0.03 | 1.05 ± 0.01 | 1.11 ± 0.01 | 1.06 ± 0.02 | 1.10 ± 0.01 | |
| 10−7 M | 1.08 ± 0.04 | 1.11 ± 0.04 | 1.05 ± 0.01 | 1.08 ± 0.01 | 1.08 ± 0.04 | 1.08 ± 0.06 | 1.06 ± 0.01 | 1.11 ± 0.01 | 1.08 ± 0.03 | 1.06 ± 0.07 | 1.06 ± 0.01 | 1.08 ± 0.06 | 1.07 ± 0.01 | |
| 3 × 10−7 M | 1.09 ± 0.04 | 1.07 ± 0.06 | 1.08 ± 0.03 | 1.09 ± 0.03 | 1.10 ± 0.01 | 1.04 ± 0.04 | 1.08 ± 0.05 | 1.07 ± 0.01 | 1.11 ± 0.01 | 1.05 ± 0.03 | 1.10 ± 0.01 | 1.09 ± 0.01 | 1.09 ± 0.03 | |
| 10−6 M | 1.08 ± 0.01 | 1.04 ± 0.04 | 1.05 ± 0.02 | 1.10 ± 0.01 | 1.12 ± 0.05 | 1.11 ± 0.04 | 1.10 ± 0.04 | 1.14 ± 0.04 | 1.04 ± 0.04 | 1.09 ± 0.08 | 1.08 ± 0.06 | 1.09 ± 0.01 | 1.12 ± 0.01 | |
| 3 × 10−6 M | 1.05 ± 0.01 | 1.12 ± 0.02 | 1.08 ± 0.02 | 1.06 ± 0.01 | 1.07 ± 0.04 | 1.06 ± 0.04 | 1.11 ± 0.01 | 1.08 ± 0.07 | 1.13 ± 0.02 | 1.10 ± 0.06 | N.T. | |||
| 10−5 M | 1.07 ± 0.01 | 1.06 ± 0.03 | 1.09 ± 0.02 | 1.10 ± 0.01 | 1.10 ± 0.01 | 1.07 ± 0.02 | 1.09 ± 0.04 | 1.14 ± 0.05 | 1.10 ± 0.05 | 1.09 ± 0.02 | N.T. | |||
| Anti-estrogenic activity+ | C+ | 6.49 ± 0.44 | 6.53 ± 0.24 | 6.47 ± 0.10 | 6.46 ± 0.12 | 6.51 ± 0.03 | 6.44 ± 0.02 | 6.59 ± 0.02 | 6.58 ± 0.03 | 6.53 ± 0.21 | 6.60 ± 0.11 | 6.62 ± 0.37 | 6.48 ± 0.13 | 6.61 ± 0.13 |
| C− | 1.00 * ± 0.01 | 1.00 * ± 0.03 | 1.00 * ± 0.10 | 1.00 * ± 0.01 | 1.00 * ± 0.02 | 1.00 * ± 0.01 | 1.00 * ± 0.11 | 1.00 * ± 0.01 | 1.00 * ± 0.03 | 1.00 * ± 0.04 | 1.00 * ± 0.01 | 1.00 * ± 0.02 | 1.00 * ± 0.01 | |
| 10−9 M | N.T. | 6.70 ± 0.09 | 6.48 ± 0.26 | 6.60 ± 0.11 | ||||||||||
| 3 × 10−9 M | N.T. | 6.55 ± 0.12 | 6.57 ± 0.06 | 6.73 ± 0.24 | ||||||||||
| 10−8 M | 6.72 ± 0.03 | 6.58 ± 0.35 | 6.43 ± 0.21 | 6.58 ± 0.01 | 6.44 ± 0.21 | 6.36 ± 0.01 | 6.61 ± 0.18 | 6.62 ± 0.37 | 6.37 ± 0.25 | 6.50 ± 0.08 | 6.64 ± 0.10 | 6.71 ± 0.06 | 6.50 ± 0.26 | |
| 3 × 10−8 M | 6.47 ± 0.01 | 6.74 ± 0.20 | 6.57 ± 0.02 | 6.53 ± 0.01 | 6.53 ± 0.21 | 6.50 ± 0.04 | 6.45 ± 0.02 | 6.76 ± 0.20 | 6.62 ± 0.26 | 6.57 ± 0.13 | 6.35 ± 0.03 | 6.32 ± 0.12 | 6.58 ± 0.19 | |
| 10−7 M | 6.59 ± 0.06 | 6.75 ± 0.11 | 6.53 ± 0.04 | 6.53 ± 0.13 | 6.49 ± 0.37 | 6.56 ± 0.03 | 6.69 ± 0.18 | 6.61 ± 0.13 | 6.67 ± 0.24 | 6.51 ± 0.08 | 6.77 ± 0.11 | 6.57 ± 0.04 | 6.53 ± 0.24 | |
| 3 × 10−7 M | 6.66 ± 0.05 | 6.77 ± 0.14 | 6.54 ± 0.23 | 6.61 ± 0.08 | 6.43 ± 0.16 | 6.45 ± 0.05 | 6.63 ± 0.16 | 6.68 ± 0.28 | 6.59 ± 0.12 | 6.37 ± 0.21 | 6.65 ± 0.17 | 6.77 ± 0.02 | 6.54 ± 0.25 | |
| 10−6 M | 6.48 ± 0.09 | 6.59 ± 0.25 | 6.54 ± 0.33 | 6.54 ± 0.33 | 6.50 ± 0.37 | 6.63 ± 0.03 | 6.60 ± 0.09 | 6.80 ± 0.17 | 6.53 ± 0.17 | 6.63 ± 0.21 | 6.65 ± 0.12 | 6.52 ± 0.04 | 6.54 ± 0.06 | |
| 3 × 10−6 M | 6.58 ± 0.09 | 6.72 ± 0.46 | 6.59 ± 0.12 | 6.59 ± 0.07 | 6.42 ± 0.17 | 6.58 ± 0.05 | 6.56 ± 0.01 | 6.54 ± 0.20 | 6.43 ± 0.18 | 6.45 ± 0.01 | N.T. | |||
| 10−5 M | 6.49 ± 0.01 | 6.60 ± 0.04 | 6.66 ± 0.22 | 6.49 ± 0.05 | 6.57 ± 0.12 | 6.62 ± 0.14 | 6.59 ± 0.09 | 6.60 ± 0.12 | 6.58 ± 0.39 | 6.64 ± 0.14 | N.T. | |||
C+: positive control (E2 1 × 10−10 M); C−: negative control (culture media); N.T.: not tested; SD: Standard deviation. *: Significant difference compared to C− (estrogenic activity) or C+ (anti-estrogenic activity) (p < 0.05). Results are expressed as MCF-7 cell proliferation (fold over control), calculated as the ratio between the maximum proliferation induced by the sample and that of the negative control (C−). +For anti-estrogenic activity, MCF-7 cells were treated with the bioprinting materials and E2 (100 pM). ABS: acrylonitrile butadiene styrene; b-TPUe: 1,4-butanediol thermoplastic polyurethane elastomer; GelMA: methacrylated gelatine; HAMA: methacrylated hyaluronic acid; PCL: polycaprolactone; PEGDA: poly(ethylene glycol) diacrylate; PLA: polylactic acid; PVA: polyvinyl alcohol.
Table 2.
Transcriptional activity of the human androgen receptor (hAR) of 13 bioprinting materials measured by the PALM assay.
| PLA | ABS | PCL | b-TPUe | PEDGA | Hyaluronic Acid | Agarose | Silk Fibroin | GelMa | HAMA | PVA | Collagen | Alginate | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Androgenic activity | C+ | 106.59 * ± 5.37 | 95.40 * ± 0.28 | 100.83 * ± 0.56 | 100.27 * ± 0.95 | 99.46 * ± 0.94 | 101.29 * ± 4.27 | 101.61 * ± 1.21 | 99.62 * ± 5.83 | 98.64 * ± 3.55 | 96.18 * ± 7.00 | 99.78 * ± 0.81 | 99.43 * ± 0.93 | 102.59 * ± 4.31 |
| C− | 10.21 ± 0.24 | 9.68 ± 0.11 | 9.81 ± 0.69 | 10.05 ± 0.07 | 10.56 ± 1.34 | 10.04 ± 0.65 | 10.00 ± 1.50 | 9.24 ± 1.13 | 9.81 ± 0.36 | 9.31 ± 0.30 | 10.64 ± 0.93 | 9.64 ± 0.87 | 9.78 ± 0.21 | |
| 10−9 M | N.T. | 10.63 ± 0.67 | 9.96 ± 0.28 | 10.16 ± 0.32 | ||||||||||
| 3 × 10−9 M | N.T. | 10.85 ± 0.76 | 10.16 ± 0.32 | 9.45 ± 0.13 | ||||||||||
| 10−8 M | 9.82 ± 1.46 | 9.06 ± 0.16 | 10.20 ± 0.72 | 10.40 ± 0.43 | 10.46 ± 0.77 | 9.86 ± 0.73 | 10.02 ± 1.35 | 9.16 ± 1.12 | 10.03 ± 0.28 | 9.35 ± 0.13 | 10.41 ± 0.77 | 10.60 ± 0.50 | 10.30 ± 0.86 | |
| 3 × 10−8 M | 9.63 ± 0.48 | 9.77 ± 0.34 | 10.38 ± 0.48 | 10.23 ± 0.38 | 10.84 ± 1.12 | 10.21 ± 0.76 | 10.16 ± 1.42 | 9.58 ± 0.84 | 9.90 ± 0.08 | 9.74 ± 0.22 | 10.47 ± 0.84 | 10.16 ± 0.51 | 9.24 ± 0.69 | |
| 10−7 M | 10.73 ± 0.95 | 9.63 ± 0.13 | 10.31 ± 0.28 | 10.39 ± 0.45 | 10.49 ± 1.24 | 10.44 ± 1.04 | 9.94 ± 1.11 | 9.86 ± 1.23 | 9.93 ± 0.56 | 9.88 ± 0.37 | 10.17 ± 1.47 | 10.22 ± 0.84 | 9.86 ± 0.32 | |
| 3 × 10−7 M | 10.11 ± 0.06 | 9.61 ± 0.92 | 10.08 ± 0.24 | 10.04 ± 0.08 | 10.66 ± 0.09 | 10.07 ± 0.71 | 9.86 ± 1.38 | 9.57 ± 0.84 | 10.23 ± 0.42 | 10.04 ± 0.38 | 10.35 ± 1.44 | 10.0 ± 0.67 | 9.83 ± 0.68 | |
| 10−6 M | 9.68 ± 1.22 | 9.66 ± 1.24 | 10.42 ± 0.89 | 9.90 ± 0.85 | 10.53 ± 1.29 | 10.11 ± 1.32 | 10.21 ± 0.59 | 9.76 ± 1.17 | 10.49 ± 0.80 | 9.46 ± 0.87 | 10.95 ± 1.67 | 10.93 ± 0.63 | 10.09 ± 0.99 | |
| 3 × 10−6 M | 10.33 ± 0.63 | 9.44 ± 0.02 | 10.13 ± 0.32 | 10.29 ± 0.43 | 10.40 ± 1.50 | 10.16 ± 0.83 | 10.47 ± 0.93 | 9.84 ± 0.58 | 9.86 ± 0.85 | 10.06 ± 0.81 | N.T. | |||
| 10−5 M | 11.14 ± 0.20 | 9.78 ± 0.65 | 10.05 ± 1.34 | 9.81 ± 0.27 | 10.29 ± 1.29 | 10.66 ± 1.76 | 11.23 ± 0.93 | 10.31 ± 1.41 | 10.61 ± 1.86 | 9.95 ± 0.87 | N.T. | |||
| Anti-androgenic activity+ | C+ | 81.79 ± 0.10 | 82.81 ± 0.45 | 81.82 ± 1.49 | 83.28 ± 1.46 | 88.14 ± 0.03 | 82.40 ± 0.89 | 80.34 ± 1.47 | 81.08 ± 1.50 | 80.91 ± 0.22 | 81.03 ± 0.05 | 78.79 ± 1.91 | 80.80 ± 0.80 | 81.18 ± 0.32 |
| C− | 10.05 * ± 0.20 | 9.95 * ± 0.23 | 10.20 * ± 0.10 | 10.11 * ± 0.14 | 10.13 * ± 0.12 | 10.11 * ± 0.21 | 9.89 * ± 0.11 | 10.25 * ± 0.25 | 9.88 * ± 0.13 | 10.02 * ± 0.08 | 10.23 * ± 0.21 | 10.33 * ± 0.12 | 9.98 * ± 0.25 | |
| 10−9 M | N.T. | 80.92 ± 0.10 | 80.88 ± 0.84 | 81.54 ± 1.02 | ||||||||||
| 3 × 10−9 M | N.T. | 79.15 ± 1.12 | 82.24 ± 1.51 | 81.80 ± 0.43 | ||||||||||
| 10−8 M | 83.72 ± 0.10 | 81.10 ± 0.45 | 83.02 ± 2.78 | 80.98 ± 1.33 | 82.28 ± 2.04 | 80.42 ± 0.84 | 81.66 ± 0.36 | 81.08 ± 0.97 | 82.45 ± 0.32 | 81.20 ± 0.48 | 81.40 ± 1.02 | 80.22 ± 1.86 | 80.17 ± 1.82 | |
| 3 × 10−8 M | 83.77 ± 0.47 | 83.03 ± 0.79 | 83.63 ± 1.39 | 84.56 ± 0.69 | 82.07 ± 0.52 | 82.73 ± 1.58 | 82.74 ± 0.34 | 83.40 ± 1.46 | 83.00 ± 1.84 | 84.21 ± 2.17 | 81.55 ± 0.84 | 81.17 ± 2.97 | 82.29 ± 1.72 | |
| 10−7 M | 81.57 ± 0.34 | 80.79 ± 1.34 | 83.71 ± 1.75 | 82.92 ± 0.03 | 82.99 ± 2.45 | 81.97 ± 1.01 | 83.49 ± 2.60 | 81.82 ± 0.28 | 82.38 ± 0.60 | 82.61 ± 0.96 | 80.90 ± 3.33 | 82.81 ± 2.24 | 79.99 ± 0.42 | |
| 3 × 10−7 M | 85.02 ± 0.48 | 82.35 ± 1.17 | 81.66 ± 1.75 | 82.59 ± 2.50 | 80.94 ± 1.03 | 81.80 ± 1.68 | 81.42 ± 0.91 | 81.76 ± 0.82 | 82.20 ± 0.24 | 82.11 ± 0.75 | 80.10 ± 2.49 | 82.06 ± 1.34 | 83.00 ± 0.44 | |
| 10−6 M | 83.54 ± 0.47 | 84.20 ± 0.90 | 83.55 ± 1.18 | 84.33 ± 2.15 | 82.82 ± 0.45 | 81.56 ± 1.46 | 83.50 ± 0.59 | 82.23 ± 1.34 | 82.76 ± 0.19 | 82.44 ± 2.40 | 81.80 ± 0.35 | 80.56 ± 0.83 | 81.51 ± 2.50 | |
| 3 × 10−6 M | 81.55 ± 0.46 | 80.76 ± 0.45 | 83.69 ± 1.86 | 82.89 ± 1.83 | 82.97 ± 2.57 | 81.72 ± 3.18 | 83.90 ± 2.81 | 82.38 ± 3.08 | 82.96 ± 1.05 | 82.06 ± 2.20 | N.T. | |||
| 10−5 M | 82.92 ± 0.46 | 80.65 ± 1.47 | 82.18 ± 2.38 | 82.76 ± 0.13 | 81.53 ± 1.76 | 82.27 ± 0.25 | 82.19 ± 1.90 | 81.68 ± 1.90 | 82.98 ± 1.23 | 81.51 ± 2.58 | N.T. | |||
C+: positive control (R1881 1 × 10−7 M); C−: negative control (culture media); N.T.: not tested; SD: Standard deviation. *: Significant difference compared to C− (androgenic activity) or C+ (anti-androgenic activity) (p < 0.05). Results are expressed as maximal luciferase activity (%), where 100% corresponds to the luciferase activity induced by 100 nM R1881. + For anti-androgenic activity, PALM cells were treated with the bioprinting materials and R1881 (0.3 nM). ABS: acrylonitrile butadiene styrene; b-TPUe: 1,4-butanediol thermoplastic polyurethane elastomer; GelMA: methacrylated gelatine; HAMA: methacrylated hyaluronic acid; PCL: polycaprolactone; PEGDA: poly(ethylene glycol) diacrylate; PLA: polylactic acid; PVA: polyvinyl alcohol.
Furthermore, MTT assays showed no significant effects on the metabolic viability of MCF-7 and PALM cells, with viability values ranging from 95% to 100% at all tested concentrations, confirming the absence of relevant cytotoxic effects (Supplementary Tables S1 and S2).
Our results indicate that, within the tested concentration ranges and experimental conditions analyzed, no detectable estrogenic, anti-estrogenic, androgenic, or anti-androgenic activity, nor significant toxicity, was observed for all the biomaterials tested. The concentrations evaluated in this study should be interpreted as screening concentrations under controlled conditions and do not necessarily reflect in vivo exposure levels. Further studies evaluating compound release from printed constructs and their long-term biological effects are required to determine the relevance of these findings under realistic exposure scenarios.
4. Conclusions
This study represents the first in vitro screening assessment of selected endocrine activities—specifically, estrogenic and anti-androgenic activities—in a wide range of materials used in 3D printing and bioprinting. The aim of this work was to provide an initial assessment, rather than a comprehensive evaluation of the endocrine-disrupting potential or overall biological safety of these materials. Under the experimental conditions and concentration ranges tested, no detectable estrogenic, anti-estrogenic, androgenic or anti-androgenic activity was observed through the estrogen and androgen receptor pathways. However, these findings should not be interpreted as definitive evidence of the complete absence of endocrine activity. Further studies addressing additional toxicological and biological parameters, as well as long-term in vitro and in vivo assessments, are required to comprehensively characterize their safety and suitability for 3D-printing and bioprinting applications.
Acknowledgments
This article is part of the PhD thesis developed by E. Nygren-Jiménez under the Biomedicine Program of the University of Granada.
Abbreviations
The following abbreviations are used in this manuscript:
| ABS | Acrylonitrile butadiene styrene |
| B-TPUe | 1,4-butanediol thermoplastic polyurethane elastomer |
| GelMA | methacrylated gelatine |
| HAMA | methacrylated hyaluronic acid |
| PCL | Polycaprolactone |
| PEGDA | poly(ethylene glycol) diacrylate |
| PLA | polylactic acid |
| PVA | polyvinyl alcohol |
| R1881 | reference androgen metiltrienolone |
Supplementary Materials
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/toxics14090760/s1, Table S1: Cell viability of MCF-7 cells exposed to 13 bioprinting materials and controls assessed by the MTT assay; Table S2: Cell viability of PALM cells exposed to 13 bioprinting materials and controls assessed by the MTT assay.
Author Contributions
A.O.-M. and E.N.-J.: Conceptualization, funding acquisition, project administration and writing—original draft; J.M.M.-M.: methodology and formal analysis; G.J.: writing—review & editing; M.F.F. and J.A.M.: Supervision, writing—review & editing. All authors have read and agreed to the published version of the manuscript.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This study was supported by the “Programa de Proyectos de Investigación Precompetitivos para Jóvenes Investigadores—Modalidades A y B” from the University of Granada (UGR) (grant no. PPJIB2023-075). The authors are also grateful to the Institute of Health Carlos III (ISCIII) for the predoctoral contract awarded to A. Olivas-Martínez (grant no. FI21/00236).
Footnotes
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
References
- 1.Leberfinger A.N., Dinda S., Wu Y., Koduru S.V., Ozbolat V., Ravnic D.J., Ozbolat I.T. Bioprinting functional tissues. Acta Biomater. 2019;95:32–49. doi: 10.1016/j.actbio.2019.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Groll J., Burdick J.A., Cho D.W., Derby B., Gelinsky M., Heilshorn S.C., Jüngst T., Malda J., Mironov V.A., Nakayama K., et al. A definition of bioinks and their distinction from biomaterial inks. Biofabrication. 2018;11:013001. doi: 10.1088/1758-5090/aaec52. [DOI] [PubMed] [Google Scholar]
- 3.Khoeini R., Nosrati H., Akbarzadeh A., Eftekhari A., Kavetskyy T., Khalilov R., Ahmadian E., Nasibova A., Datta P., Roshangar L., et al. Natural and synthetic bioinks for 3D bioprinting. Adv. NanoBiomed Res. 2021;1:2000097. doi: 10.1002/anbr.202000097. [DOI] [Google Scholar]
- 4.Peter E., Monisha J., George S.A. Bisphenol-A release from thermoplastic clear aligner materials: A systematic review. J. Orthod. 2023;50:276–286. doi: 10.1177/14653125231160570. [DOI] [PubMed] [Google Scholar]
- 5.Tichy A., Simkova M., Schweiger J., Bradna P., Güth J.F. Release of bisphenol A from milled and 3D-printed dental polycarbonate materials. Materials. 2021;14:5868. doi: 10.3390/ma14195868. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Boonen I., De Nys S., Vervliet P., Covaci A., Van Landuyt K.L., Duca R.C., Godderis L., Denison M.S., Elskens M. Assessing the estrogenic activity of chemicals present in resin based dental composites and in leachates of commercially available composites using the ERα-CALUX bioassay. Dent. Mater. 2021;37:1834–1844. doi: 10.1016/j.dental.2021.09.013. [DOI] [PubMed] [Google Scholar]
- 7.Fernández M.F., Olea N. Disruptores endocrinos, ¿suficiente evidencia para actuar? Gac. Sanit. 2014;28:93–95. doi: 10.1016/j.gaceta.2013.11.005. [DOI] [PubMed] [Google Scholar]
- 8.Olivas-Martínez A., Ventura-Wischner P.S., Fernandez M.F., Freire C. Influence of exposure to endocrine disruptors and other environmental chemicals on breast development in girls: A systematic review of human studies. Int. J. Hyg. Environ. Health. 2025;263:114487. doi: 10.1016/j.ijheh.2024.114487. [DOI] [PubMed] [Google Scholar]
- 9.Cichos S., Schätzlein E., Wiesmann-Imilowski N., Blaeser A., Henrich D., Frank J., Drees P., Gercek E., Ritz U. A new 3D-printed polylactic acid-bioglass composite for bone tissue engineering induces angiogenesis in vitro and in ovo. Int. J. Bioprinting. 2023;9:751. doi: 10.18063/ijb.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Xiao L., Shimamura N., Kamio T., Ide R., Mochizuki M., Nakahara T. Polycarbonate–acrylonitrile butadiene styrene three dimensional printing material exhibits biocompatibility and enhances osteogenesis and gingival tissue formation with human cells. Cells. 2025;14:167. doi: 10.3390/cells14030167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Koch F., Thaden O., Conrad S., Tröndle K., Finkenzeller G., Zengerle R., Kartmann S., Zimmermann S., Koltay P. Mechanical properties of polycaprolactone (PCL) scaffolds for hybrid 3D-bioprinting with alginate-gelatin hydrogel. J. Mech. Behav. Biomed. Mater. 2022;130:105219. doi: 10.1016/j.jmbbm.2022.105219. [DOI] [PubMed] [Google Scholar]
- 12.Martínez-Moreno D., Jiménez G., Chocarro-Wrona C., Carrillo E., Montañez E., Galocha-León C., Clares-Naveros B., Gálvez-Martín P., Rus G., De Vicente J., et al. Pore geometry influences growth and cell adhesion of infrapatellar mesenchymal stem cells in biofabricated 3D thermoplastic scaffolds useful for cartilage tissue engineering. Mater. Sci. Eng. C. 2021;122:111933. doi: 10.1016/j.msec.2021.111933. [DOI] [PubMed] [Google Scholar]
- 13.Martínez-Moreno D., Venegas-Bustos D., Rus G., Gálvez-Martín P., Jiménez G., Marchal J.A. Chondro-Inductive b-TPUe-Based Functionalized Scaffolds for Application in Cartilage Tissue Engineering. Adv. Healthc. Mater. 2022;11:2200251. doi: 10.1002/adhm.202200251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Ahmed S., Hussain R., Khan A., Batool S.A., Mughal A., Nawaz M.H., Irfan M., Wadood A., Avcu E., Rehman M.A.U. 3D printing assisted fabrication of copper–silver mesoporous bioactive glass nanoparticles reinforced sodium alginate/poly (vinyl alcohol) based composite scaffolds: Designed for skin tissue engineering. ACS Appl. Bio Mater. 2023;6:5052–5066. doi: 10.1021/acsabm.3c00726. [DOI] [PubMed] [Google Scholar]
- 15.He B., Wang J., Xie M., Xu M., Zhang Y., Hao H., Xing X., Lu W., Han Q., Liu W. 3D printed biomimetic epithelium/stroma bilayer hydrogel implant for corneal regeneration. Bioact. Mater. 2022;17:234–247. doi: 10.1016/j.bioactmat.2022.01.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Kutlehria S., Dinh T.C., Bagde A., Patel N., Gebeyehu A., Singh M. High-throughput 3D bioprinting of corneal stromal equivalents. J. Biomed. Mater. Res. Part B Appl. Biomater. 2020;108:2981–2994. doi: 10.1002/jbm.b.34628. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Duarte Campos D.F., Rohde M., Ross M., Anvari P., Blaeser A., Vogt M., Panfil C., Yam G.H.F., Mehta J.S., Fischer H., et al. Corneal bioprinting utilizing collagen-based bioinks and primary human keratocytes. J. Biomed. Mater. Res. Part A. 2019;107:1945–1953. doi: 10.1002/jbm.a.36702. [DOI] [PubMed] [Google Scholar]
- 18.Di Buduo C.A., Lunghi M., Kuzmenko V., Laurent P.A., Della Rosa G., Del Fante C., Nogare D.E.D., Jug F., Perotti C., Eto K., et al. Bioprinting Soft 3D Models of Hematopoiesis using Natural Silk Fibroin-Based Bioink Efficiently Supports Platelet Differentiation. Adv. Sci. 2024;11:2308276. doi: 10.1002/advs.202308276. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Sasikumar S.C., Goswami U., Raichur A.M. 3D bioprinting with visible light cross-linkable mucin-hyaluronic acid composite bioink for lung tissue engineering. ACS Appl. Bio Mater. 2024;7:5411–5422. doi: 10.1021/acsabm.4c00579. [DOI] [PubMed] [Google Scholar]
- 20.Bhutani U., Dey N., Chowdhury S.K., Waghmare N., Mahapatra R.D., Selvakumar K., Chandru A., Bhowmick T., Agrawal P. Biopolymeric corneal lenticules by digital light processing based bioprinting: A dynamic substitute for corneal transplant. Biomed. Mater. 2024;19:035017. doi: 10.1088/1748-605X/ad3312. [DOI] [PubMed] [Google Scholar]
- 21.Lee G.W., Chandrasekharan A., Roy S., Thamarappalli A., Mahaling B., Lee H., Seong K.Y., Ghosh S., Yang S.Y. 3D bioprinting of stromal cells-laden artificial cornea based on visible light-crosslinkable bioinks forming multilength networks. Biofabrication. 2024;16:035002. doi: 10.1088/1758-5090/ad35eb. [DOI] [PubMed] [Google Scholar]
- 22.Molina-Molina J.M., Amaya E., Grimaldi M., Sáenz J.M., Real M., Fernández M.F., Balaguer P., Olea N. In vitro study on the agonistic and antagonistic activities of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear receptors. Toxicol. Appl. Pharmacol. 2013;272:127–136. doi: 10.1016/j.taap.2013.05.015. [DOI] [PubMed] [Google Scholar]
- 23.Molina-Molina J.M., Real M., Jiménez-Díaz I., Belhassen H., Hedhili A., Torné P., Fernández M.F., Olea N. Assessment of estrogenic and anti-androgenic activities of the mycotoxin zearalenone and its metabolites using in vitro receptor specific bioassays. Food Chem. Toxicol. 2014;74:233–239. doi: 10.1016/j.fct.2014.10.008. [DOI] [PubMed] [Google Scholar]
- 24.Denizot F., Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods. 1986;89:271–277. doi: 10.1016/0022-1759(86)90368-6. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.