Engineering a 3D wounded skin equivalent to study early inflammatory and regenerative responses in vitro.

INTRODUCTION: Traditional models for studying wound healing, including 2D cell cultures and animal models, present substantial limitations in mimicking human skin physiology. In this study, we present a three-dimensional wounded skin equivalent (3DWoundSE) composed of human cells as a physiologically relevant in vitro platform to investigate wound healing processes. METHODS: The model builds upon a previously established 3D skin equivalent (3DSE) and incorporates a reproducible partial-thickness dermal punch wound. We characterised the 3DWoundSE using histology, cytotoxicity assays, immunofluorescence staining, and pro-inflammatory cytokine profiling at multiple time points post-wounding. RESULTS: Results revealed hallmark wound responses, including increased lactate dehydrogenase (LDH) and apoptosis-inducing factor (AIF) expression, dynamic Ki-67 proliferation changes, and a pro-inflammatory cytokine response, notably elevated IL-6, IL-8, IL-33 and TNF-α levels. DISCUSSION: Compared to the intact 3DSE, this 3DWoundSE demonstrated enhanced responsiveness to injury and cytotoxic stimuli, confirming its utility for early wound response assessment. This platform offers a reproducible and ethically sound alternative to animal models, with potential applications in dermatological research, drug development, and therapeutic screening.