Proteome and microbiome profiles of polymicrobial salivary biofilms on 3D MEW fibrous scaffolds: biomimetic ECM-inspired structures

Replicating the structural complexity of polymicrobial oral biofilms <i>in vitro</i> remains a significant challenge in biomaterials research. Nevertheless, developing clinically relevant biofilm models is crucial for advancing our understanding of biofilm-host interactions and elucidating how biomaterials influence microbial composition, behaviour, and overall biofilm dynamics. In this work, 3D biomimetic fibrous scaffolds made from medical-grade polycaprolactone (mPCL) were fabricated using the melt electrowriting (MEW) technique. The effects on biofilm viability, activity, microbiome, and proteome profiles were assessed on 3D fibrous scaffolds and conventional 2D tissue culture plates (TCP). Human saliva was cultured on MEW mPCL (3D BF) and TCP (2D BF) for 4 days, followed by microbiome profiling <i>via</i> 16S rRNA sequencing and proteomic analysis using LC-MS/MS, SWATH with GO and KEGG pathway enrichment. The results demonstrated that 3D MEW mPCL scaffolds enhanced biofilm biomass, thickness, and viability. Microbiome analysis revealed that 3D BF was enriched with both commensals and pathogens, including <i>Veillonella, Peptostreptococcus, Porphyromonas gingivalis, and Treponema denticola</i>, alongside probiotic species like <i>Lactobacillus acidophilus</i>. Pooled proteomic data from three technical repeats, along with GO and KEGG analyses, revealed a functionally dynamic biofilm ecosystem characterised by elevated expression of proteins involved in glycolysis, the TCA cycle, and nucleotide metabolism, highlighting pathways essential for biofilm survival, stress adaptation, and host interaction. These 'proof-of-concept' findings highlight the potential of 3D MEW fibrous mPCL scaffolds as a biomimetic 3D platform capable of accurately recapitulating the dynamic spatial and metabolic complexity of oral biofilms, thereby facilitating innovative investigations into microbial ecology, host-pathogen interactions, and the accelerated development of targeted antimicrobial therapies.