Engineered nanotopography on electrospun microfibers alters cytokine production in polarized macrophages

Abstract

The goal of this work is to engineer the surface structure of aligned, electrospun fibers known to direct axonal extension in order to alter macrophage polarization. Electrospun fibers were engineered to have nanoscale surface pits of different size and density on the surface of the fibers depending on the amount of non-solvent that was added to the electrospinning solution. Mechanical testing of electrospun fibers with nanoscale surface pits had a 60% increase in elastic modulus compared to fibers of identical chemical composition but a smooth surface topography. Primary mouse bone marrow macrophages (BMMs) were cultured on electrospun microfibers with specific nanoscale surface structures and given an inflammatory stimulus (LPS, M1), a regenerative stimulus (LPS + immune complexes, M2) or no stimulus (M0). For M1 polarized macrophages, transcription of IL-12 production (an M1 inflammatory cytokine) was reduced in one group (fibers with surface divets) compared to all other groups with different nanotopography, but protein expression was only significantly different between fibers with divets versus fibers with a large number of nanoscale pits. IL-10 production (an M2 regenerative cytokine) was not affected by any surface structures at either the RNA or protein levels. Inducible nitric oxide synthase (iNOS) was not reduced for any group except for electrospun fibers with a smooth surface, and NO end products in the supernatant confirmed the changes observed at the RNA level.

The similarities in fiber rigidity from mechanical tests of the fibers suggest fiber rigidity cannot completely account for the differences in cytokine production in inflammatory macrophages, but the mechanical properties may have a synergistic effect with nanoscale topography. Taken together, it is concluded from these results that fiber nanotopography can be engineered using phase separation principles, the elastic modulus of a fiber can be drastically increased by addition of nanoscale pits, and fiber nanotopography may alter inflammatory cytokine production in inflammatory macrophages.

Macrophages are highly dynamic immune cells involved in both inflammation and repair. In spinal cord injury, inflammatory (M1) macrophages cause axotomy and dystrophic bulbs that cause further damage to neurons and ultimately prevent regeneration. Recent interest in tissue engineering focuses on the design of biomaterials that can modify macrophage polarization by changing the material shape or mechanical properties (i.e. stiffness).