Biocompatibility of 3D-Printed Methacrylate for Hearing Devices

The capacity of 3D printing (3DP) technologies to initiate speedy polymerization of solvent-free resins accounts for their utility in the manufacturing of medical devices. Nonetheless, independent biological evaluation of 3D-printed materials is recommended due to the unique parameters of the manufacturing process, which can influence their physical, chemical and biological properties. In this study, E-Shell 450 clear methacrylate indicated for 3DP of hearing devices was examined for biological safety using zebrafish bioassays adapted to Organization for Economic Cooperation and Development (OECD) fish embryo test. In addition, the proprietary material was characterized for composition using headspace gas chromatography–mass spectrometry (GC–MS). To initiate the biological test, newly fertilized zebrafish eggs were cultured on non-treated and ethanol-treated methacrylates in glass petri dishes with ultrapure water, incubated at 28.5 ◦C and assessed for developmental endpoints of toxicity at 24 h intervals until 96 h. Toxicological data indicate that non-treated methacrylate is extremely toxic in zebrafish bioassays, whereas ethanol-treated counterpart showed a relative lower toxicity possibly due to ethanoic–aqueous interactions as observed by GC–MS. With the current influx of 3D printing materials, users are urged to exercise caution. Operators must also take cognizance of the potential toxicity of the chemicals used in 3DP and implement safety measures to limit their exposure.


Introduction
The recent hype surrounding 3D printing (3DP) attests to its growing popularity in almost every manufacturing sector including medicine, architecture, sports, aerospace and automotive engineering and contemporary arts [1].3DP comprise a host of processes and technologies that offer a diverse spectrum of capabilities for the manufacturing of end-use products and devices in different materials [2].The digital manufacturing process simply involves feeding a virtual model (usually 'STL' file) into a designated 3D printer to build parts in successive layers until the desired 3D part is completed.Despite the potentials of 3DP offering significant benefits in terms of speed [3], independent biological evaluation of manufactured devices is highly recommended [4,5] due to the unique parameters of the manufacturing process, which can influence their physical, chemical and biological properties [6].In this study, E-Shell 450 clear methacrylate indicated for hearing devices is examined for biological safety using zebrafish bioassays adapted to the Organization for Economic Cooperation and Development (OECD) fish embryo test [7].The zebrafish is considered an excellent model for developmental toxicity, offering economy and ease of quantifying multiple toxicity endpoints [8], as well as fulfilling the pertinent aim to replace, reduce or refine the use of animals for the purposes of research or hazard identification [9,10].Representative materials were built with Inventions 2018, 3, 52 2 of 7 digital light processing (DLP) technology [11].DLP is similar to stereolithography (SL) in that both are vat photopolymerization processes that require washing built parts in organic solvents to remove any wet resin remnants, followed by postcuring to harden them.However, DLP uses a more conventional light source such as an arc lamp, with a liquid crystal display panel or a deformable mirror device, which is applied to the entire surface of the vat of resin in a single pass, relatively making it faster than SL [2].To extrapolate toxicity effects to residual monomer and degradation products [12] that may be present in the proprietary material, it was characterized for chemical composition using headspace gas chromatography-mass spectrometry (GC-MS).

Materials and Methods
EnvisionTec GmbH (Brüsseler Str.51, 45968 Gladbeck, Germany) supplied 60 × 3 mm disk-shaped samples that were built from E-Shell 450 clear resin.Postcuring (2 × 100 flashes) of samples was completed by the manufacturer in Otoflash G171 (NK-Optik GmbH, Isarstr.2, D-82065 Baierbrunn, Germany).Table 1 shows the manufacturing parameters, hazardous composition [13] and physical properties [14] of photocured E-Shell 450 clear (Figure 1).digital light processing (DLP) technology [11].DLP is similar to stereolithography (SL) in that both are vat photopolymerization processes that require washing built parts in organic solvents to remove any wet resin remnants, followed by postcuring to harden them.However, DLP uses a more conventional light source such as an arc lamp, with a liquid crystal display panel or a deformable mirror device, which is applied to the entire surface of the vat of resin in a single pass, relatively making it faster than SL [2].To extrapolate toxicity effects to residual monomer and degradation products [12] that may be present in the proprietary material, it was characterized for chemical composition using headspace gas chromatography-mass spectrometry (GC-MS).

Results
After 24 h, non-treated methacrylate induced ≈70% embryo death or lethality while surviving embryos (Figure 2) were largely unhealthy, hence test was discontinued.Although ethanol-treated methacrylate recorded only 5% mortality after 24 h, additional 50% with cumulative hypopigmentation, pericardial edema, yolk sac resorption delay and hypoactive behavior were observed in surviving fish by 96 h (Figure 3).Average growth length in surviving fish after 96 h was 3241.30µm compared to 3590.33 µm in controls.At the end of the test, fish were euthanized in 0.4% anaesthetic tricaine mesylate solution.

Results
After 24 h, non-treated methacrylate induced ≈70% embryo death or lethality while surviving embryos (Figure 2) were largely unhealthy, hence test was discontinued.Although ethanol-treated methacrylate recorded only 5% mortality after 24 h, additional 50% with cumulative hypopigmentation, pericardial edema, yolk sac resorption delay and hypoactive behavior were observed in surviving fish by 96 h (Figure 3).Average growth length in surviving fish after 96 h was 3241.30μm compared to 3590.33 μm in controls.At the end of the test, fish were euthanized in 0.4% anaesthetic tricaine mesylate solution.

Qualitative Gas Chromatography-Mass Spectrometry
Since the toxicological effects observed in bioassays are likely due to residual monomer and degradation products, the photocured materials were examined for chemical composition using headspace GC-MS.Prior to analysis, they were frozen in liquid nitrogen at −196 °C and ground into powder before being tested in Shimadzu TQ8040 GC-MS/MS (Shimadzu Corporation, Tokyo, Japan).GC column is Agilent J&W DB5-MS 30 m 0.25 mm ID 0.25 μm film thickness.Test parameters were column oven temperature at 40.0 °C, injection temperature at 250 °C, column flow rate at 1.16 mL/min, split ratio of 5.0 and a total run time of 15 min.GC-MS data shown in Table 3 shows 16 chemical compounds that reduced to 5 with the applied ethanol treatment.For reliability, only chemical compounds observed in ≥75% or ≥ = 3/4 of the samples are reported.

Qualitative Gas Chromatography-Mass Spectrometry
Since the toxicological effects observed in bioassays are likely due to residual monomer and degradation products, the photocured materials were examined for chemical composition using headspace GC-MS.Prior to analysis, they were frozen in liquid nitrogen at −196 • C and ground into powder before being tested in Shimadzu TQ8040 GC-MS/MS (Shimadzu Corporation, Tokyo, Japan).GC column is Agilent J&W DB5-MS 30 m 0.25 mm ID 0.25 µm film thickness.Test parameters were column oven temperature at 40.0 • C, injection temperature at 250 • C, column flow rate at 1.16 mL/min, split ratio of 5.0 and a total run time of 15 min.GC-MS data shown in Table 3 shows 16 chemical compounds that reduced to 5 with the applied ethanol treatment.For reliability, only chemical compounds observed in ≥75% or ≥ = 3/4 of the samples are reported.

Discussion
Experimental results in this study indicate that non-treated methacrylate is extremely toxic in zebrafish bioassay.Ethanol-treated methacrylate, on the other hand, induced a relatively lower lethality but surviving fish showed cumulative sublethal and teratogenic effects.The improved biological performance is likely due to induced swelling in polymeric chains, which allowed chemical compounds to diffuse in the ultrapure water [18] used to rinse the samples.
Acrylics are probably the most versatile family of monomers that can be used to prepare polymers with rigid, flexible, ionic, nonionic, hydrophobic or hydrophilic properties [19].They are preferred in free-radical synthesis of plastics because of the high reactivity of the acrylate double bond.Under intense ultraviolet illumination, cross-linking polymerization of liquid resins proceeds extensively within a fraction of a second to generate a 3D polymer network [20].As per standard definitions [21], E-Shell 450 is a surface device, hence it does not require stringent biological evaluation compared to methacrylates for intraoral devices.Nonetheless, uncured methacrylate monomers can be absorbed through the skin [22] and cause allergic reactions (e.g., dermatitis) [23,24].In addition, the toxicity of methacrylate esters is theorized to involve alkylation of critical cellular nucleophiles via Michael addition [25].Some of the developmental endpoints observed in toxic bioassays are comparable to those reported in animal studies that linked methacrylic esters to embryonic fetal toxicity, teratogenicity [22] and cardiovascular function [26,27].The observed chemical compounds are also used in industrial applications and can be toxic to humans if present in threshold dose.For instance, cyclomethicone is used in cosmetic and personal products [28], 2-hydroxyethyl methacrylate for desensitizing teeth, benzaldehyde for pharmaceutical products and texanol as fuel additives [29].It is worth emphasizing that inhalation toxicity [30] may result from exposure to liquid photopolymer resins, which are often characterised by unpleasant odour.For enhanced manufacturing outcomes, resins should possess high curing rate, good storage stability, low toxicity, low viscosity, and display adequate mechanical properties after photocuring [31].Resins with relatively low viscosity are likely to produce rapid polymerisation yielding cross-linked polymers with properties suited to the demands imposed by the target application [32].Some 3D printers have in-built heating mechanism for achieving the desired resin viscosity [33] but often not applicable to third-party materials.In general, the mechanical properties of photocured materials depend primarily on the chemical structure, functionality and concentration of the various constituents of the resin, and the degree of cure [34].

Conclusions
With the current influx of 3D printers and materials [35], it is imperative that the biological performance of 3D-printed medical devices is not overlooked.Users are advised to exercise caution and if necessary demand approved certification for the materials.Since 3DP is not a "one-stop" manufacturing process, operators should take cognizance of the potential toxicity of the chemicals used and implement safety measures to limit their exposure.The limitations of the study lie in the extrapolation of the toxicological data to human responses, hence quantitative analysis of the observed compounds and their throughput in zebrafish bioassays are recommended for further study.

Figure 1 .
Figure 1.Surface topography of E-Shell 450 clear.Imaging was carried out with Olympus AX70 Fluorescence Microscope; Monochrome FViewII Peltier cooled digital camera (Olympus, Tokyo, Japan) and running analysis software (Soft Imaging Solutions, Münster, Germany).

Figure 1 .
Figure 1.Surface topography of E-Shell 450 clear.Imaging was carried out with Olympus AX70 Fluorescence Microscope; Monochrome FViewII Peltier cooled digital camera (Olympus, Tokyo, Japan) and running analysis software (Soft Imaging Solutions, Münster, Germany).

Figure 2 .
Figure 2. Toxicity effects induced by non-treated methacrylate in zebrafish embryo bioassays after 24 h (A): CE coagulated embryo, DE dead embryo, MF malformed embryo without somite and UD underdeveloped embryo as compared to healthy embryos in controls (B).

Figure 2 .
Figure 2. Toxicity effects induced by non-treated methacrylate in zebrafish embryo bioassays after 24 h (A): CE coagulated embryo, DE dead embryo, MF malformed embryo without somite and UD underdeveloped embryo as compared to healthy embryos in controls (B).

Figure 3 .
Figure 3. Fish in toxic (A) and control (B) bioassays after 96 h.Note the phenotype differences in unhealthy zebrafish (A): HM Head malformation, HP Hypopigmentation, SC Spinal curvature, PE Pericardial edemata, YD Yolk sac resorption delay, as compared to normal zebrafish (B).

Figure 3 .
Figure 3. Fish in toxic (A) and control (B) bioassays after 96 h.Note the phenotype differences in unhealthy zebrafish (A): HM Head malformation, HP Hypopigmentation, SC Spinal curvature, PE Pericardial edemata, YD Yolk sac resorption delay, as compared to normal zebrafish (B).

Table 1 .
Manufacturing parameters, composition and physical properties of E-Shell 450 methacrylate.

Table 1 .
Manufacturing parameters, composition and physical properties of E-Shell 450 methacrylate.
at 24 h intervals until 96 h using Olympus MVX10 Research Macro Zoom Microscope, Olympus DP 72 digital colour microscope camera and cellSens imaging software (Olympus Soft Imaging Solutions GmbH, Münster, Germany).As per OECD test guidelines, fish is considered dead if one of the lethal endpoints is present.Ethical approval (MARP/2015/094, Approved date 8 September 2015) to use zebrafish embryos was issued by the Animal Ethics Committee in Monash University.

Table 2 .
Biomarkers of lethality, sublethality and teratogenicity assessed at 24 h intervals until 96 h.
at 24 h intervals until 96 h using Olympus MVX10 Research Macro Zoom Microscope, Olympus DP 72 digital colour microscope camera and cellSens imaging software (Olympus Soft Imaging Solutions GmbH, Münster, Germany).As per OECD test guidelines, fish is considered dead if one of the lethal endpoints is present.Ethical approval (MARP/2015/094, Approved date 8 September 2015) to use zebrafish embryos was issued by the Animal Ethics Committee in Monash University.

Table 2 .
Biomarkers of lethality, sublethality and teratogenicity assessed at 24 h intervals until 96 h.

Table 3 .
Chemical composition of non-treated and ethanol-treated photocured E-Shell 450 methacrylates.

Table 3 .
Chemical composition of non-treated and ethanol-treated photocured E-Shell 450 methacrylates.