Multi-wall carbon nanotubes (MWCNT) are widely used in many biomedical applications due to their excellent physical and chemical characteristics. However, the applications of MWCNT also raise health concerns. The small size and light weight of MWCNT allow them to enter the working environment as particulate matter of respirable sizes. Animal studies have shown that MWCNT cause inflammation, thickening and stiffening of the lung tissue, which leads to pulmonary diseases that are fatal and incurable. Therefore, understanding the effects of MWCNT on lung mechanics and pulmonary toxicity is important to the health safety in the applications involving MWCNT. However, due to the high cost and low throughput of the animal models, the progress in this field is very slow.
The bio-microelectromechanical systems (bio-MEMS) technology allows realistic modeling of 3D human tissue and high throughput testing of many samples in a single device, thus provides a potentially viable solution to screen the pathogenic factors. Here we report the development of a 3D human lung microtissue array device to evaluate the impact of two different types of MWCNT on lung mechanics and pulmonary toxicity.
Our device consists of arrays of PDMS microwells with each microwell containing a self-assembled microtissue hanging between a pair of micropillars. Using this device, we measured the changes in the mechanical properties and biomarkers related to the fibrogenesis in BEAS-2B human bronchial epithelial cells-populated microtissues. MWCNT (10-50um in length) and short-MWCNT (0.2-2um in length) with carboxyl modification (short-MWCNT-COOH) were selected. The cytotoxic effects of these 2 MWCNT were first evaluated by cell growth measurement and the reactive oxygen species (ROS) assay. BEAS-2B cells were treated with MWCNT at different concentrations (0, 50ng/mL, and 5ug/mL). At 48h post treatment, all treated groups showed the inhibition of cellular growth and generated significant ROS stress.
Next, the self-assembled BEAS-2B microtissues were used to study the biomechanical impacts of MWCNT. Almost all of the microtissues formed nice dog-bone shape between the micropillars and produced substantial contraction force at day 1 after initial seeding, except for the 5ug/ml short-MWCNT-COOH group, which was likely due to the cytotoxicity caused by overdose treatment of the short-MWCNT-COOH. The contraction forces continued to increase for all groups over a 3-day culture period and the 50ng/mL short-MWCNT-COOH treatment showed the largest contraction force and tissue width. However, the contraction force and tissue width of the 50ng/mL MWCNT treatment were closed to the untreated control group.
MicroRNA-21, a small non-coding RNA, has been found to mediate fibrogenic activation of lung fibrosis through the TGF-b signaling pathway, and thus represents a good biomarker for fibrogenic potential evaluation. Results from qRT-PCR showed significant up-regulation of microRNA-21 in 50ng/mL short-MWCNT-COOH treated BEAS-2B microtissues, which agreed with the high contraction force observed previously. Similarly, significant down-regulation of microRNA-21 was observed in 5ug/ml short-MWCNT-COOH treated microtissues, which was consistent with our contraction force results and probably due to the extensive cytotoxicity caused by such high concentration.
The 3D microtissue array device mimicked in vivo environment and allowed in-situ collection of mechanical properties of the model tissue under different treatment conditions. We demonstrated its application in investigating the effects of MWCNT on lung tissue mechanics and pulmonary toxicity. The applications of microtissue array may be extended to high throughput screening of potential pathogenic factors induced by pathogens and toxic materials.