UC Irvine

Engineered stone (ES) is a composite material consisting of crystalline silica, pigment, and binding resins, and has become one of the most common materials used in kitchen and bathroom countertops, alongside natural stones such as marble and granite. The rise in popularity of ES in countertops is due to its low maintenance and customizable design; however, fabricating ES countertops generates respirable particles that pose an occupational inhalation hazard to workers. ES particle exposure has been linked to silicosis, a progressive and irreversible fibrotic lung disease resulting from inhalation of crystalline silica. When silica reaches the alveoli, macrophages attempt to clear the particles through phagocytosis while initiating inflammatory responses. Persistent inflammation and cellular injury can promote fibroblasts activation and collagen production, contributing to lung fibrosis. Few studies examine how silica content in ES influences the development of silicosis. This study aims to address this gap by utilizing an in vitro respiratory model to examine cellular responses after exposure to ES particles with varying silica content and to investigate mechanisms relevant to ES-related silicosis.An in vitro co-culture system consisting of three rat cell lines, REL-6TN alveolar type II (AT2) cells, NR8383 alveolar macrophages, and RFL-6 alveolar fibroblasts, were used in this study to mimic aspects of the lung microenvironment. This model was assembled in a Transwell system, with AT2 cells and macrophages in the apical compartment and fibroblasts in the basolateral compartment. Particles generated from four commercially available ES slabs with low (< 40%; sample A and B) or high (> 40%; samples C and D) silica content, along with pure silica (Min-U-Sil 5), were used for exposure. AT2 cells and macrophages were exposed at 0, 1, 2.5, and 5 µg/cm2 via an air-liquid interface (ALI) system. Particle size distribution, z-average, and polydispersity index (PDI) in suspension were assessed by dynamic light scattering (DLS). Cells and cell culture media were collected at 24, 48, and 72 hours after exposure for subsequent analyses of cytotoxicity, inflammatory responses, and fibroblast activation. Barrier integrity of AT2 cells was also assessed through 96 hours post-exposure.At the concentration range used in this study, ES samples exhibited minimal cytotoxicity. At the highest concentration tested (5 µg/cm2 ), AT2 cells showed a significant decline in barrier integrity after exposure to samples A and D. For the inflammatory profile, samples B and C predominantly induced innate pro-inflammatory cytokines, while sample D induced a distinct cytokine profile that included mediators associated with type 2 immune responses. Additionally, sample D triggered the strongest effect in fibroblast activation. Sample C and Min-U-Sil 5 elicited moderate activation, while samples A and B induced minimal effects. In conclusion, the results demonstrated sample-specific patterns of cellular responses that were not consistently associated with the silica content of the ES samples. These findings imply that silica content alone may not predict the biological activity of ES particles and that other particle characteristics or constituents, together with silica, may contribute to cellular responses relevant to ES-related silicosis.