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

## TL;DR

A new in vitro model simulates acute and chronic wound healing using human cells and synthetic substrates, allowing testing of wound dressings and healing mechanisms.

## Contribution

The first in vitro vascularized dermis model that mimics acute and chronic wound conditions using a synthetic biomimetic substrate and macrophage polarization.

## Key findings

- PhenoDrive-Y promotes 3D tissue-like structures with fibroblasts and endothelial cells on 2D culture plates.
- U932 monocytes differentiate into M1 and M2 phenotypes depending on wound conditions simulated.
- Chronic wound-like conditions show impaired angiogenesis and disorganized extracellular matrix compared to acute wounds.

## Abstract

This work presents for the first time a novel in vitro model of wound healing where immortalized human fibroblast and vascular endothelial cell are driven to form vascularized tissue-like structures by the synthetic biomimetic substrate PhenoDrive-Y and subsequently studied for their ability to reform and close the gap created by a conventional scratching step. The addition of U937 monocytes without or with the spiking of pro-inflammatory cytokine enables the simulation of the early phases of acute and chronic wound conditions. The model was validated here for its potential to assess the biocompatibility of two clinically available wound dressings; one based on cellulose engineered in the form of tulle (N-A Ultra) and the other made of alginate hydrogel (Kaltostat).

What are the main findings?
The organotypic culture of human fibroblasts and vascular endothelial cells on PhenoDrive-Y led to the formation of 3D tissue-like structures on a conventional 2D tissue culture plate.The monocytic U937 cell lines are shown to differentiate into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes by contact with PhenoDrive-Y, albeit differing if acute and chronic wound-like conditions were adopted.The conventional scratch test was taken to a new level where the formation of vascularized tissue-like structures rather than simple fibroblast monolayer formation in acute and chronic conditions was optimized.

The organotypic culture of human fibroblasts and vascular endothelial cells on PhenoDrive-Y led to the formation of 3D tissue-like structures on a conventional 2D tissue culture plate.

The monocytic U937 cell lines are shown to differentiate into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes by contact with PhenoDrive-Y, albeit differing if acute and chronic wound-like conditions were adopted.

The conventional scratch test was taken to a new level where the formation of vascularized tissue-like structures rather than simple fibroblast monolayer formation in acute and chronic conditions was optimized.

What are the implications of the main findings?
The in vitro model, simulating the early phases of high inflammatory burden, enables the identification of the effect of acute and chronic conditions on macrophage differentiation in either the M1 pro-inflammatory or M2 post-inflammatory phenotype of wound healing, as well as the different secretory profiles of extracellular matrix components.The presence of wound dressings of different physicochemical properties can, to some extent, change the above profiles, leading to different rates of wound healing.

The in vitro model, simulating the early phases of high inflammatory burden, enables the identification of the effect of acute and chronic conditions on macrophage differentiation in either the M1 pro-inflammatory or M2 post-inflammatory phenotype of wound healing, as well as the different secretory profiles of extracellular matrix components.

The presence of wound dressings of different physicochemical properties can, to some extent, change the above profiles, leading to different rates of wound healing.

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.