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

Abstract

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.

Calcium phosphate coating enhances osteointegration of melt electrowritten scaffold by regulating macrophage polarization

“…2 D). The phenomenon might be attributed to the fact that cells displayed different shapes in both the 2D and 3D growth patterns [ 55 ]. It seemed that RAW264.7 cells adhered to the scaffold grew in a 2D manner because the scaffold provided a surface for cell attachment and the subsequent protrusion of pseudopodia in various configurations.…”

Structure–Activity Relationship (SAR) of Flavones on Their Anti-Inflammatory Activity in Murine Macrophages in Culture through the NF-κB Pathway and c-Src Kinase Receptor

“…This is also why some studies have reached completely divergent conclusions at the cellular level and the living animal level . Though three-dimensional (3D) cell culture models have been employed to mimic the extracellular matrix (ECM) for anti-inflammatory drug screening and the mechanisms of their activity on inflammatory disease, there are still rare 3D cell models applied to flavones. , Recently, our group utilized poly­(ε- caprolactone) (PCL) to establish a 3D RAW 264.7 macrophage model to more accurately mimic in vivo conditions and screen anti-inflammatory agents, which could be applied to draw SAR of flavones in the future. Additionally, animal and clinical studies should be conducted to provide in vivo and clinical evidence for SAR and promote the application of flavones as an anti-inflammatory agent in the future.…”

Antioxidant and Anti-Inflammatory Effects of 6,3’,4´- and 7,3´,4´-Trihydroxyflavone on 2D and 3D RAW264.7 Models

“…These misleading results in in vitro examinations could be partially attributed to the divergent difference between the predominate plate-cultured and actual tissue cells. The 3D model was proven to close the gap between in vitro and in vivo examinations, minimize false positive results, and provide a reliable prediction of efficacy through better recapitulation of in vitro cell biology and growth microenvironment [13]. Therefore, the two flavones showing pro-inflammatory-mediator-suppressive activity on 2D and, especially, 3D macrophages, indicated their potential application as natural anti-inflammatory agents.…”

“…Electrohydrodynamic (EHDJ)-printed poly(ε-caprolactone) (PCL) scaffolds are biocompatible, lowly immunogenic, optimally biodegradable, and have comparable pore and fiber size (approximately 10 µm) matching macrophage scale [12]. The 3D macrophages show higher lipopolysaccharide (LPS) sensitivity and lower antiinflammatory agent sensitivity, delivering higher physiological relevance [13].…”