LB1784 Novel scaffold-free 3D skin equivalent to fully replicate human dermal remodeling and epithelialization during wound healing

Restoration of skin after injuries is crucial for life and involves extensive communication between the different cellular constituents of the diverse compartments of the skin and its extracellular matrix (ECM). Numerous models of wound healing have been developed in recent years and are usually restricted to conventional 2D monolayer format. To date, most of the existing 3D bioengineered wound healing models are mainly based on the use exogenous of non-human scaffold failing in replicating the modifications of the dermal ECM during the closure process. Here, we developed a novel 3D bilayered wound healing model consisting of a 3D dermal equivalent made up entirely of human fibroblasts embedded in their own ECM, on which epidermal cells were seeded. After 42 days of culture, this scaffold-free 3D skin equivalent was injured with a biopsy punch, creating a 3 mm wound that extended through both epidermis and dermis. The wounded region was investigated for up to 12 days in terms of re-epithelialization and dermal remodeling by using histological and immunohistological analysis. During the culture period, we observed a complete re-epithelization and closure of the defect. The epidermis regenerated progressively from the surrounding wound margins. After 3 days, a migrating tongue of collectively migrating keratinocytes was detected starting from the differentiated preexisting epithelium across the wound. At day 7, the differentiation of the neoepidermis and the regeneration of the dermal-epidermal junction were more advanced than toward the wound center, where the proliferative index was significantly higher. At the dermal level, we observed concomitantly an ECM remodeling with the expression of collagens, fibronectin and hyaluronan. Application of healing stimulators significantly accelerated the healing process suggesting the responsiveness of the model to stimuli. Thus, this novel scaffold-free 3D wound healing model provides a new tool to capture complex wound healing mechanics as well as to evaluate treatments applied to wounds.