Engineered Nanotopography on the Microfibers of 3D-Printed PCL Scaffolds to Modulate Cellular Responses and Establish an In Vitro Tumor Model

Abstract

Scaffold-based three-dimensional (3D) cell culture systems have gained increased interest in cell biology, tissue engineering, and drug screening fields as a replacement of twodimensional (2D) monolayer cell culture and as a way to provide biomimetic extracellular matrix environments. In this study, microscale fibrous scaffolds were fabricated via electrohydrodynamic printing, and nanoscale features were created on the fiber surface by simply leaching gliadin of poly(ε-caprolactone) (PCL)/ gliadin composites in ethanol solution. The microstructure of the printed scaffolds could be precisely controlled by printing parameters, and the surface nanotopography of the printed fiber could be tuned by varying the PCL/gliadin ratios. By seeding mouse embryonic fibroblast (NIH/3T3) cells and human nonsmall cell lung cancer (A549) cells on the printed scaffolds, the cellular responses showed that the fiber nanotopography on printed scaffolds efficiently favored cell adhesion, migration, proliferation, and tissue formation. Quantitative analysis of the transcript expression levels of A549 cells seeded on nanoporous scaffolds further revealed the upregulation of integrin-β1, focal adhesion kinase, Ki-67, E-cadherin, and epithelial growth factor receptors over what was observed in the cells grown on the pure PCL scaffold. Furthermore, a significant difference was found in the relevant biomarker expression on the developed scaffolds compared with that in the monolayer culture, demonstrating the potential of cancer cell-seeded scaffolds as 3D in vitro tumor models for cancer research and drug screening.

Three-Dimensional RAW264.7 Cell Model on Electrohydrodynamic Printed Poly(ε-Caprolactone) Scaffolds for In Vitro Study of Anti-Inflammatory Compounds

“…2 This differs from their natural growth manner where spherical macrophages are supported by neighboring cells and ECM 27 and thus lead to false positive anti-inflammatory compound screening results. 7,8 EHDJ is a highly precisely controlled 3D printing technology, and the EHDJ-printed scaffolds have been applied to culture various cell lines, for example, NIH/3T3 and A549 cells on PCL and PCL/zein scaffolds, 11,26 murine macrophages on NIR-II-dye-containing PCL scaffolds, 28 and human macrophages and PC12 cells on PCL scaffolds. 22,29 In all these studies, cells showed behaviors different from traditional in vitro cells and demonstrated their potential of biomimetic structural organization and application in tissue engineering.…”

“…Among various techniques, electrohydrodynamic jetting (EHDJ) is an emerging technology that precisely controls pore size, geometry, and fiber diameter of scaffolds. 11 It combines the working principles of the additive manufacturing technique and the electrospinning technique. On the one hand, it applies a stage motion and layer-by-layer stacking principle in additive manufacturing to fabricate scaffolds with certain pattern and 3D structure, which is also the reason for it to belong to 3D printing approaches; on the other hand, it applies the main principle in electrospinning.…”

“…Over the past 20 years, 3D cell models attracted increasing interest in cell biology to bridge the gap between in vitro 2D cell culture and in vivo tissues because they can better mimic a physiological environment and provide predictive data for in vivo studies. , The 3D cell models were first and extensively applied in cancer studies, and the results found that they have a more persistent proliferation period, spheroid shape and multilayer morphology, more ECM within aggregates, higher differentiation ability, and different genes and protein expression levels. − More importantly, 3D cell models could closely match tumor xenografts in a genome-scale CRISPR screen and in anti-cancer drug screening. , Consequently, 3D cell culture methods have been developed particularly in tissue engineering and regenerative medicine, and so far endothelial cells, neural cells, fibroblasts, primary bone mesenchymal stem cells, and other stems cells have been cultured in a 3D manner to solving implantation and wound healing problems. Given the in vivo biomimetic advantage, 3D cell models are essential for new generation of cell cultures for cell biology and precise drug screening.…”

“…Recent progress in additive manufacturing techniques enables 3D scaffolds’ construction. Among various techniques, electrohydrodynamic jetting (EHDJ) is an emerging technology that precisely controls pore size, geometry, and fiber diameter of scaffolds . It combines the working principles of the additive manufacturing technique and the electrospinning technique.…”

“…This closely mimics the scale of living cells and ECM. In our previous study, poly­(ε-caprolactone) (PCL) scaffolds were printed on a home-built EHDJ printer, and human lung cancer cell A549 and murine embryonic fibroblast NIH/3T3 cells successfully proliferated on the scaffolds. , The microscale PCL scaffolds with ultrafine fibers (∼10 μm) and highly defined geometries closely resemble the scale of living cells and ECM and thus exhibit potential as a 3D cell model . Consequently, herein, we devised a 3D murine macrophage model on EHDJ-printed PCL scaffolds for studying anti-inflammatory activity of pharmaceutical agents and natural products.…”