A dispersal-recolonisation 3D biofilm in vitro model based on co-assembled peptide amphiphiles and clinical wound fluid

Chronic wound infections are sustained by dynamic three-dimensional (3D) biofilm cycles involving maturation, dispersal, and recolonisation, yet existing in vitro models fail to reproduce these temporal and structural complexities. Here, we report a strategy that co-assembles a designed protease-inhibitory peptide amphiphile (PA) with patient-derived wound fluid (WF) to reconstruct the complete biofilm life cycle in vitro. The PA sequence incorporates an HWGF motif capable of binding and inhibiting matrix metalloproteinase-9 (MMP-9), thereby preserving the integrity of recolonised biofilms under proteolytic stress. Co-assembling with WF generated a living material that mimicked the biochemical and mechanical microenvironment of chronic wounds, supporting the formation of stable 3D biofilms capable of dispersal and recolonisation. Furthermore, we established a controllable polymicrobial infection model and benchmarked its antibiotic-response trends against conventional susceptibility testing and in vivo spatial microbiological analyses. Notably, the antibiotic-response patterns of the PA/WF-derived biofilms closely mirrored those observed in a rat wound infection in vivo model. Collectively, our findings demonstrate that co-assembled living materials can recapitulate the nutritional composition, 3D architecture, and recolonisation dynamics of in vivo infectious biofilms, offering a physiologically relevant and customisable platform for investigating chronic wound infections and accelerating anti-biofilm drug discovery.