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

Inflammation plays an essential role in the human immune system, and anti-inflammatory compounds are important to promote health. However, the in vitro screening of these compounds is largely dependent on flat biology. Herein, we report our efforts in establishing a 3D inflammation murine macrophage model. Murine macrophage RAW 264.7 cells were cultured on poly(ε-caprolactone) (PCL) scaffolds fabricated through an electrohydrodynamic jetting 3D printer and their behavior were examined. Cells on PCL scaffolds showed a 3D shape and morphology with multilayers and a lower proliferation rate. Moreover, macrophages were not activated by scaffold material PCL and 3D microenvironment. The 3D cells showed greater sensitivity to lipopolysaccharide stimulation with higher production activity of nitric oxide (NO), nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2). Additionally, the 3D macrophage model showed lower drug sensitivity to commercial anti-inflammatory drugs including aspirin, ibuprofen, and dexamethasone, and natural flavones apigenin and luteolin with higher IC50 for NO production and lower iNOS and COX-2 inhibition efficacy. Overall, the 3D macrophage model showed promise for higher accurate screening of anti-inflammatory compounds. We developed, for the first time, a 3D macrophage model based on a 3D-printed PCL scaffold that provides an extracellular matrix environment for cells to grow in the 3D dimension. 3D-grown RAW 264.7 cells showed different sensitivities and responses to anti-inflammatory compounds from its 2D model. The 3D cells have lower sensitivity to both commercial and natural anti-inflammatory compounds. Consequently, our 3D macrophage model could be applied to screen anti-inflammatory compounds more accurately and thus holds great potential in next-generation drug screening applications.

Zein Increases the Cytoaffinity and Biodegradability of Scaffolds 3D-Printed with Zein and Poly(ε-caprolactone) Composite Ink.

Electrohydrodynamic printing (EHDP) has attracted extensive interests as a powerful technology to fabricate micro- to nano-scale fibrous scaffolds in a custom-tailored manner for biomedical applications. A few synthetic biopolymer inks are applicable to this EHDP technology, but the fabricated scaffolds suffered from low mechanical strength, biocompatibility, and biodegradability. In this study, a series of poly(ε-caprolactone) (PCL)/zein composite inks were developed and their printability was examined on a solution-based EHDP system for scaffold fabrication. Multilayer grid scaffolds were manufactured by PCL, PCL/zein-10, and PCL/zein-20 inks, respectively and characterized. The mechanical strength of scaffolds printed by PCL/zein composite inks was remarkably enhanced in terms of Young's modulus and yield stress. The enzyme-accelerated in vitro degradation study demonstrated that zein-containing scaffolds exhibited dose-responsive improvement on the degradation rate as evidenced by surface morphological change of fibers. Moreover, the biocompatibility of PCL/zein scaffolds, tested on mice embryonic fibroblast (NIH/3T3) and human nonsmall lung cancer cell (H1299), manifested better cell affinity. Our findings suggest that scaffolds fabricated by the solution-based EHDP with PCL/zein composite inks can significantly improve Young's modulus, yield stress, biocompatibility, and biodegradability and have potential applications in drug delivery systems, 3D cell culture modeling, or tissue engineering.