Fabrication and characterization of a 3D polymicrobial microcosm biofilm model using melt electrowritten scaffolds

The majority of current biofilm models or substrates are two-dimensional (2D) and support biofilm growth in the horizontal plane only. Three-dimensional (3D) substrates may support both horizontal and vertical biofilm growth. This study compared biofilm growth quantity and quality between highly porous 3D micrometric fibrous scaffolds and 2D film substrates fabricated from medical grade polycaprolactone (mPCL). Melt electrowriting (MEW), a high-resolution additive manufacturing technology, was employed to design orderly aligned fine (~12 μm) fibre-based 3D scaffolds, while 2D films were fabricated by a casting method. The 3D scaffolds with a controlled pore size of 100 and 250 μm and thickness of ~0.8 mm and 2D films were incubated in pooled saliva collected from six volunteers for 1, 2, 4, 7 and 10 days at 37 °C to facilitate polymicrobial biofilm formation. Crystal violet assay demonstrated greater biofilm biomass in 3D MEW scaffolds than in 2D films. Biofilm thickness in 3D scaffolds was significantly higher compared to the biofilm thickness in 2D films. Both biovolume and substratum coverage of the biofilms was higher in the 3D scaffolds compared to 2D films. Polymeric bridges, pores, and channels characteristic of biofilms could be demonstrated by scanning electron microscopy. 16S rRNA sequencing demonstrated that the polymicrobial biofilms in the 3D scaffolds were able to retain 60-70 % of the original inoculum microbiome after 4 days. The MEW-fabricated 3D fibrous scaffold is a promising substrate for supporting multidirectional biofilm growth and modelling of a polymicrobial microcosm.