A Novel In Vitro Vascularized Dermis Organotypic Model of Acute and Chronic-Like Wounds.

Acute and chronic wounds are a major clinical burden, with persistent inflammation, impaired fibroblast function, defective angiogenesis, and disordered extracellular matrix deposition. The translational potential of existing in vitro models is limited by their poor durability and physiological relevance. The present paper aims to develop a robust in vitro organotypic model to simulate the early phases of both acute and chronic wounds and to validate it by testing the biocompatibility of clinically available wound dressings. Human fibroblasts and vascular endothelial cell lines were cultured at a ratio of 1:1 for 48 h, either on uncoated tissue culture plastic or on tissue culture plastic coated with a synthetic substrate (PhenoDrive-Y) that biomimics the extracellular matrix and promotes cell organization into tissue-like structures on a 2D plane (i.e., angiogenesis sprouting and fibroblast organization around it). Wound conditions were then created by damaging the formed structures using a conventional scratch procedure and introducing U937 human macrophage cells to the model to simulate either the onset of an acute wound or that of a chronic wound through the simultaneous spiking of the culture with relevant cytokines, i.e., IL-6 and TNF-α. The formation of new tissue-like structures in the scratch area was quantified by the extent of scratch closure after a further 24 h of incubation. Morphological analysis of wound healing was performed by light microscopy, while angiogenesis was assessed by CD31 immunostaining by confocal microscopy. The deposition of components of the extracellular matrix was determined both qualitatively and quantitatively by Picrosirius Red staining for collagen production and by Alcian Blue staining for glycosoaminoglycan synthesis on the adhering cells and their supernatants. Macrophage polarization into either M1 or M2 phenotype was studied by immunostaining with iNOS (M1) and CD206 (M2) antibodies by confocal microscopy. The model was validated by studying the gap closure areas in simulated acute and chronic wound-like conditions when incubated with clinically available wound dressings, N-A Ultra and Kaltostat. PhenoDrive-Y allowed the formation of tissue-like structures on the 2D tissue culture plane as opposed to the formation of cell monolayers on the uncoated tissue culture plastic. Upon mechanical damage, cell migration was significantly different; uncoated control co-cultures achieved complete closure as an indistinct monolayer by 24 h, while the organotypic wound models showed a slower percentage of damage closure. A further delay in the closure of the damaged area was observed when chronic wound-like conditions were simulated. Angiogenesis in chronic wound conditions was considerably impaired compared to the acute conditions. The analysis of the extracellular matrix component synthesis, specifically collagen and polysaccharides, revealed the deposition of dense, organized collagen fibers in the acute wound model, in contrast to the thin, fragmented collagen fibers and intracellular polysaccharides observed under chronic wound-like conditions. This corresponded to a statistically significant increase in the levels of both collagen and polysaccharides detected as soluble molecules in the supernatants. Macrophage polarization showed no statistically significant differences in the acute and chronic wound models, though iNOS did significantly decrease after N-A application in acute and chronic models. However, acute wound-like conditions showed a restoration of the vascularized tissue-like structures after treatment with these types of dressings, albeit through different organizational pathways, whereas only minimal improvement was noted under chronic wound conditions, particularly in the case of the N-A dressing. The organotypic dermis model for the onsets of acute and chronic wounds emerges as a highly versatile tool to understand healing mechanisms in the absence or presence of co-morbidities and to assess the biocompatibility of wound dressings as well as the safety, efficacy and dosage of drugs.