Engineering a 3D Wounded Skin Equivalent to Study Early Inflammatory and Regenerative Responses In Vitro

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 <i>in vitro</i> platform to investigate wound healing processes. The model builds upon a previously established 3D skin equivalent (3DSE) and incorporates a reproducible full-thickness central wound. We characterised the 3DWoundSE using histology, cytotoxicity assays, immunofluorescence staining, and pro-inflammatory cytokine profiling at multiple time points post-wounding. Results revealed hallmark wound responses, including increased 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. 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.

传统的伤口愈合研究模型（包括二维细胞培养和动物模型）在模拟人体皮肤生理特征方面存在显著局限性。本研究中，我们提出一种由人类细胞构成的三维损伤皮肤等效模型（3DWoundSE），作为具有生理相关性的<i>in vitro</i>平台，用于探究伤口愈合过程。该模型基于先前建立的三维皮肤等效模型（3DSE），并整合了可重复的全层中央伤口。我们通过组织学、细胞毒性测定、免疫荧光染色以及损伤后多个时间点的促炎细胞因子谱分析，对3DWoundSE进行了表征。结果显示出典型的伤口反应，包括乳酸脱氢酶（LDH）和凋亡诱导因子（AIF）表达升高、Ki-67增殖的动态变化，以及促炎细胞因子反应（尤其是IL-6、IL-8、IL-33和TNF-α水平显著升高）。与完整的3DSE相比，该3DWoundSE对损伤和细胞毒性刺激表现出增强的响应性，证实了其在早期伤口反应评估中的实用性。此平台为动物模型提供了可重复且伦理上合理的替代方案，在皮肤病学研究、药物开发和治疗筛选方面具有潜在应用价值。