Assessing Leachable Cytotoxicity of 3D-Printed Polymers and Facile Detoxification Methods.

Additive manufacturing of polymers is gaining momentum in health care industries by providing rapid 3D printing of customizable designs. Yet, little is explored about the cytotoxicity of leachable toxins that the 3D printing process introduced into the final product. We studied three printable materials, which have various mechanical properties and are widely used in stereolithography 3D printing. We evaluated the cytotoxicity of these materials through exposing two fibroblast cell lines (human and mouse derived) to the 3D-printed parts, using overlay indirect contact assays. All the 3D-printed parts were measured toxic to the cells in a leachable manner, with flexible materials more toxic than rigid materials. Furthermore, we attempted to reduce the toxicity of the 3D-printed material by employing three treatment methods (further curing, passivation coating, and Soxhlet solvent extraction). The Soxhlet solvent extraction method was the most effective in removing the leachable toxins, resulting in the eradication of the material's toxicity. Passivation coating and further curing showed moderate and little detoxification, respectively. Additionally, mechanical testing of the materials treated with extraction methods revealed no significant impacts on its mechanical performances. As leachable toxins are broadly present in 3D-printed polymers, our cytotoxicity evaluation and reduction methods could aid in extending the selections of biocompatible materials and pave the way for the translational use of 3D printing.

Versatile activity and morphological effects of zinc oxide submicron particles as anticancer agents.

Background: Submicron particles (SMPs), as novel bionanomaterials, offer complementary benefits to their conventional nano-counterparts. Aim: To explore zinc oxide (ZnO) SMPs' bioimaging and anticancer potentials. Materials & methods: ZnO SMPs were synthesized into two shapes. Fluorescent spectrum and microscopy were studied for the bioimaging property. Wound healing and Live/Dead assays of glioblastoma cells were characterized for anticancer activities. Results: ZnO SMPs exhibited a high quantum yield (49%) with stable orange fluorescence emission. Both morphologies (most significant in the rod shape) showed tumor-selective properties in cytotoxicity, inhibition to cell migration and attenuating the cancer-upregulated genes. The tumor selectivity was attributed to particle degradation and surface properties on pH dependency. Conclusion: The authors propose that ZnO SMPs could be a promising anticancer drug with tunable, morphology-dependent properties for bioimaging and controlled release.

Submicron particles (SMPs) are a novel nanomaterial whose total size is microscale (around one-millionth of a meter), but at least one dimension is nanoscale (around one-billionth of a meter). Their combined micro- and nanoscale properties are complementary, which can be an improvement on their conventional nano-counterparts. The aim of this study was to explore the bioimaging and anticancer properties of zinc oxide (ZnO) SMPs. ZnO SMPs were synthesized in two shapes: rod-shaped and flower-shaped. The fluorescence spectra and microscopy images were studied to investigate their potential for imaging applications, and wound healing and viability assays of glioblastoma cells were used to characterize anticancer activity. ZnO SMPs exhibited strong and stable orange fluorescence emission. Both shapes of ZnO SMPs showed tumor-selective toxicity, inhibition to cell migration and attenuating the cancer-upregulated genes; however, these effects were more significant for the rod-shaped particles. The tumor selectivity was attributed to pH-dependent particle degradation related to surface properties. The authors therefore propose that ZnO SMPs could be a promising anticancer drug with tunable, morphology-dependent properties for bioimaging and controlled release.

A matrigel-free method to generate matured human cerebral organoids using 3D-Printed microwell arrays.

The current methods of generating human cerebral organoids rely excessively on the use of Matrigel or other external extracellular matrices (ECM) for cell micro-environmental modulation. Matrigel embedding is problematic for long-term culture and clinical applications due to high inconsistency and other limitations. In this study, we developed a novel microwell culture platform based on 3D printing. This platform, without using Matrigel or external signaling molecules (i.e., SMAD and Wnt inhibitors), successfully generated matured human cerebral organoids with robust formation of high-level features (i.e., wrinkling/folding, lumens, neuronal layers). The formation and timing were comparable or superior to the current Matrigel methods, yet with improved consistency. The effect of microwell geometries (curvature and resolution) and coating materials (i.e., mPEG, Lipidure, BSA) was studied, showing that mPEG outperformed all other coating materials, while curved-bottom microwells outperformed flat-bottom ones. In addition, high-resolution printing outperformed low-resolution printing by creating faithful, isotropically-shaped microwells. The trend of these effects was consistent across all developmental characteristics, including EB formation efficiency and sphericity, organoid size, wrinkling index, lumen size and thickness, and neuronal layer thickness. Overall, the microwell device that was mPEG-coated, high-resolution printed, and bottom curved demonstrated the highest efficacy in promoting organoid development. This platform provided a promising strategy for generating uniform and mature human cerebral organoids as an alternative to Matrigel/ECM-embedding methods.

Fabrication of Tapered 3D Microstructure Arrays Using Dual-Exposure Lithography (DEL).

Three-dimensional (3D) microstructure arrays (MSAs) have been widely used in material science and biomedical applications by providing superhydrophobic surfaces, cell-interactive topography, and optical diffraction. These properties are tunable through the engineering of microstructure shapes, dimensions, tapering, and aspect ratios. However, the current fabrication methods are often too complex, expensive, or low-throughput. Here, we present a cost-effective approach to fabricating tapered 3D MSAs using dual-exposure lithography (DEL) and soft lithography. DEL used a strip-patterned film mask to expose the SU-8 photoresist twice. The mask was re-oriented between exposures (90° or 45°), forming an array of dual-exposed areas. The intensity distribution from both exposures overlapped and created an array of 3D overcut micro-pockets in the unexposed regions. These micro-pockets were replicated to DEL-MSAs in polydimethylsiloxane (PDMS). The shape and dimension of DEL-MSAs were tuned by varying the DEL parameters (e.g., exposure energy, inter-exposure wait time, and the photomask re-orientation angle). Further, we characterized various properties of our DEL-MSAs and studied the impact of their shape and dimension. All DEL-MSAs showed optical diffraction capability and increased hydrophobicity compared to plain PDMS surface. The hydrophobicity and diffraction angles were tunable based on the MSA shape and aspect ratio. Among the five MSAs fabricated, the two tallest DEL-MSAs demonstrated superhydrophobicity (contact angles >150°). Further, these tallest structures also demonstrated patterning proteins (with ~6-7 µm resolution), and mammalian cells, through microcontact printing and direct culturing, respectively. Our DEL method is simple, scalable, and cost-effective to fabricate structure-tunable microstructures for anti-wetting, optical-, and bio-applications.