In vitro and in vivo toxicological evaluation of emissions from the fused filament fabrication three-dimensional printing

Author

Mariana T. FarcasFollow

Semester

Spring

Date of Graduation

2021

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Yon Rojanasakul

Committee Co-Chair

Yong Qian

Committee Member

Vincent Castranova

Committee Member

Timothy R. Nurkiewicz

Committee Member

Aleksandr B. Stefaniak

Abstract

Fused filament fabrication (FFF), a three-dimensional (3-D) printing process, is an emerging technology that has recently gained wide popularity among both consumers and manufacturers. As filament is heated to above its glass transition temperature in a 3-D printer, a portion may undergo thermal decompostion, which releases ultrafine particles (UFP) and volatile organic compounds (VOCs) with potential adverse respiratory health effects are released into the air. This study's central hypotheses is that emissions generated during 3-D printing are toxic and exposure to these emissions induces pulmonary and systemic adverse health effects. Considering that currently, limited understanding is available on the health impact of the FFF 3-D printer exposures, the overall goal of this research was to fill the knowledge gap by pursuing the following three studies.

The aim of the first study was to assess acrylonitrile butadiene styrene (ABS) and polycarbonate (PC) filaments 3-D printer emissions-induced cell toxicity. In this study, the particles and vapors released during printing were collected directly into the cell culture medium and delivered to the small airway epithelial cells at six concentrations. At 24 h, various endpoints, such as cellular uptake, cell viability, cell membrane damage, ROS production, total antioxidant capacity, glutathione peroxidase levels in cell lysates, cell death mechanisms (apoptosis and necrosis), and cytokines and chemokines released in cell supernatants were measured. To analyze the data, mixed model regression analyses were performed on these endpoints using particle numbers as the independent variable. The regression lines illustrated a significant doseresponse relationship between a decrease in cell viability and the number of emitted particles, which correlated with a significant dose-dependent increase in the LDH activity, and all other endpoints evaluated.

The aim of the second study was to assess pulmonary and systemic toxicity in rats following whole-body inhalation exposure to ABS filament 3-D printer emissions. In this study, male Sprague Dawley rats were exposed to filtered air or ABS emissions for 1, 4, 8, 15, or 30 days (4 h/day, 4 days/week). The average mass concentration and number of particulate generated during 4-h real-time printing of three desktop 3-D printers operating simultaneously was 240 µg/m3 and 8.84 x 10⁴ particles/m³. At the start of the exposure (day 1), a predominant pro-inflammatory response was seen in BALF, represented by an increase in IFN-γ and TNF-α Th1-type cytokines followed by a switch to an anti-inflammatory response by day 15 of exposure represented by a rise in IL-10 Th2-type cytokine. The Th1/Th2 switch could be responsible for the initial “delayed” influx of the alveolar macrophages and its peak occurrence at 15 days of exposure, which corresponded with a significant increase in blood monocytes and platelet counts. Other systemic changes noted were that initially (day 1), a significant increase in both hepatic and renal biomarkers was found; however, at day 15 of exposure, only renal biomarkers were increased. At the longest exposure duration (day 30), all the endpoints evaluated returned to the control levels. Neither pulmonary oxidative stress responses nor histopathological changes of the lungs and nasal passages were found among the treatments.

The aim of the third study was to evaluate the pulmonary effects of FFF 3-D printer emissions using a relevant human ALI organotypic airway tissue model to mimic the respiratory behavior upon exposure. Primary normal, human-derived bronchial epithelial cells (NHBEs) were directly exposed for 4 h to ABS filament emissions. NHBEs epithelium integrity and differentiation changes, cytotoxicity, tissue injury, and inflammatory and immune system regulation markers were evaluated following exposure and 24 h after the end of the exposure. Overall, at the conditions applied, exposure of NHBEs to ABS emissions did not affect epithelium integrity, ciliation, mucus production, or induce cytotoxicity. At 24 h after the exposure, significant increases in IL-12p70, IFN-γ, TNF-α, IL-17A, VEGF, and MIP-1α were noted in the basal cell culture medium of ABS-exposed cells compared to chamber control cells.

Overall, toxicological evaluation of FFF 3-D printer emissions was conducted in three different conditions, 1) traditional submerged culture of human small airway epithelial cells, 2) repeated whole-body inhalation exposure of rats to freshly generated aerosols, and 3) advanced human ALI organotypic airway tissue model. At the experimental conditions applied, in both in vitro models and in vivo, 3-D printer emissions produced minimal to moderate pulmonary toxicity. Furthermore, these findings were consistent with results observed in the physiologically relevant in vivo-like in vitro model cultured at ALI. In conclusion, these studies indicate that the FFF 3-D printer emissions could induce moderate toxicological effects. These studies are significant as they are amongst the first and comprehensive studies published to evaluate the pulmonary and systemic toxicity of 3-D printer emissions. Further studies are needed to establish a more broad exposure-dose-response relationships and integration of in vivo and in vitro responses.

Recommended Citation

Farcas, Mariana T., "In vitro and in vivo toxicological evaluation of emissions from the fused filament fabrication three-dimensional printing" (2021). Graduate Theses, Dissertations, and Problem Reports. 8106.
https://researchrepository.wvu.edu/etd/8106

Embargo Reason

Publication Pending