P35 Nanoscale biophysics of the AP-1 pathway in wound healing

Abstract Introduction and aims Skin scarring and dysfunctional healing affect millions annually. Alterations in the collagen-rich extracellular matrix (ECM) and persistent fibroblast activation significantly impact skin regeneration, and AP-1 modulation during wound repair is a key pathway in this process. Here, we demonstrate combining small-angle X-ray scattering (SAXS) (ultrastructural collagen fibril prestrain and alignment) and nanoindentation (NI) (local indentation modulus) reveal how the nanoscale matrix mechanisms in the ECM are modulated depending on AP-1 inhibition or activation. Methods Full-thickness punch wounds were performed on adult C57BL/6 mice (five biological replicates), treated intraperitoneally with AP-1 inhibitor (T5224) or AP-1 induction (clomipramine). Ten-day wound tissue (inhibition) and 21-day wound tissue (activated) were optimal cutting temperature-embedded and cryosectioned. Slide-mounted 12-µm sections were indented (Optics11 Chiaro) to extract indentation modulus (10-µm spacing) and Ultralene-mounted thick sections were scanned using SAXS (I22 Diamond Light Source) to extract collagen concentration, fibrillar prestrain, direction and alignment (20-µm spacing). Results SAXS analysis of the AP-1 inhibitor model, compared with controls, revealed reduced collagen fibril alignment and organization, along with lower and more uniform fibrillar prestrain. Additionally, the stiffness of AP-1 inhibited wounds was reduced compared with controls. In contrast, the AP-1 induced wounds exhibited a marked increase in stiffness across the entire wound bed region. The high resolution of the analysis allows spatial mapping of the mechanical forces in the wound bed in comparison with the cellular configuration and nanoscale structural composition. Conclusions We found that AP-1 inhibition promotes wound healing by facilitating uniform fibrillar prestrain and reducing stiffness, while AP-1 induction increases local stiffness, indicative of enhanced fibroblast proliferation and scarring potential. By comparing the biomechanical and biostructural maps of AP-1-affected wound beds at the nanoscale, we can dissect the ECM dynamics at unprecedented resolution and demonstrate how structural changes within the ECM influence cellular behaviour and drive biomechanical alterations.