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.

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.

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Full text

1. Introduction

Wound healing is an exquisitely complex cascade of biochemical events controlling the activities of inflammatory and tissue cells. Indeed, the whole process of healing involves a highly synchronized and tightly regulated sequence of four overlapping phases: hemostasis, inflammation, proliferation, and tissue remodeling [1]. By definition, the healing of acute wounds, such as those induced by trauma or surgery, progresses predictably and in a timely manner through this regenerative process to achieve the structural and functional integrity of the skin within days or weeks, depending on the extent of the injury and individual physiological conditions [2]. On the contrary, chronic wounds, which are affected by co-morbidities such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers, do not follow this systematic progression [3]; they are instead characterized by being perpetually trapped in an unresolved, chronic, dysregulated inflammatory state [4]. The global impact of chronic wound epidemiology represents a major and escalating public health challenge affecting millions of individuals worldwide across all demographic groups [5,6]. The increasing rates of underlying co-morbidities such as Type 2 diabetes and cardiovascular diseases will inevitably lead to an increasing number of patients affected by chronic wounds [7,8]. Diabetic foot ulcers alone affect approximately 15% to 25% of all people suffering from diabetes at some point in their lives [9] and are the direct cause of over 80% of all non-traumatic lower extremity amputations performed worldwide [10]. Beyond severe morbidity experienced by patients, the care of these non-healing wounds places a significant burden on the healthcare system through both direct and indirect costs [11]. In developed nations, conservative annual estimates of expenditures dedicated solely to chronic wound care amount to tens of billions of dollars [12], a financial burden that critically strains public health budgets and demands more cost-effective therapeutic strategies [13].

The chronic wound microenvironment differs biochemically and cellularly from that of an acute wound. One fundamental feature of the non-healing state is a persistent imbalance between matrix-degrading enzymes and their inhibitors [14]. More precisely, chronic wounds have hyper-elevated levels of matrix metalloproteinases (MMPs) [15]. This excess of proteolytic activity results in the continuous and uncontrolled degradation of vital growth factors, receptors, and the newly deposited extracellular matrix (ECM), thus ‘trapping’ the wound in a cycle of breakdown [16]. Chronic wounds are also strongly associated with cellular senescence, particularly in the resident fibroblast population [17]. Such senescent fibroblasts display reduced proliferative capacity and impaired migratory function, along with diminished ability to synthesize and organize new collagen, all of which are fundamental processes required for successful tissue repair. The persistent presence of pro-inflammatory cytokines, including high concentrations of Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α), maintains the inflammatory loop by altering the inflammatory phenotype of cells such as macrophages; for this reason, chronic wounds are unable to transition into the proliferative phase [18,19].

Considering the complex pathophysiology of chronic wounds, an urgent need in wound-healing research is the development of standardized in vitro models that accurately reflect their complex biochemical and cellular microenvironment, thereby facilitating the study of underlying biological pathways as well as the testing of novel therapeutics and wound dressings [20,21]. Current models, mainly based on the overly simplistic scratch test of a cell monolayers or on unnecessarily complex 3D gel encapsulation systems, are unable to capture the conditions characteristic of chronic wounds or relate them effectively to acute conditions, thereby limiting advances in fundamental knowledge and hindering the rapid translation of novel therapeutic agents into clinical practice [22]. In particular, the 2D scratch assay, which is useful for initial cell migration and proliferation screening, does not provide data about cell-to-cell and cell-to-matrix interactions that play a key role in tissue repair [23]. The 3D models of cell encapsulation are typically performed using natural hydrogels, such as Matrigel or collagen [24]. The major limitation of these materials is their batch-to-batch variability and their crucial, very fast proteolytic degradation; for example, Matrigel usually loses any structural integrity after just 48 h of culture [25]. This rapid breakdown is seriously limiting longer-term observations necessary for the analysis of chronic wound modeling [26]. Therefore, the creation of a robust, well-defined, and architecturally suitable wound model that can closely resemble the dynamic and interconnected healing stages over prolonged periods of time continues to be the primary challenge [27]. The most significant and widely recognized gap in the field is the absence of a standardized, reproducible, operator-friendly and durable in vitro platform able to closely simulate and maintain the distinct conditions of acute and chronic wounds over a clinically relevant time frame [22]. Such a versatile model would be highly valuable for comparative mechanistic studies and for the efficient high-throughput screening of potential pharmaceutical interventions aimed at shifting the chronic state toward a healing trajectory. The present study aims to achieve most of these objectives by reproducing the early phases of acute and chronic wound conditions through a dermis organotypic model based on the co-culture of tissue cells responsible for contraction (fibroblasts), together with those initiating tissue vascularization (endothelial cells). The ability of these cells to form vascularized tissue-like structures upon physical damage and under monocyte challenge was used to simulate the inflammatory events occurring at the onset of healing. Spiking the model with the pro-inflammatory cytokines TNFα and IL-6 was performed to simulate the chronic state. Overall, the operator-friendly nature of this model provides an opportunity to dissect the histochemical and biochemical parameters that arise during the early phases of healing, as well as to test wound dressings and novel therapeutics.

2. Materials and Methods

2.1. Preparation of the Dermis-Like Organotypic Model

PhenoDrive-Y (PD-Y, Tissue Click Ltd., Brighton and Hove, UK) was prepared and applied as a coating to the plastic surface of tissue culture plates (TCP) according to the manufacturer’s instructions. One milliliter of 75/25% v/v ethanol/deionized water solution was added to a vial of the PD-Y containing 1 mg of freeze-dried product powder. The substrate solution was rapidly obtained by a pipetting action and transferred to a 15 mL tube (Corning, Flintshire, UK), and its volume was adjusted to 10 mL with the same 75/25% v/v ethanol/deionized water medium. Standard 24-well TCPs (Corning, Flintshire, UK) were coated by pipetting 200 µL of the prepared PD-Y solution in each well, allowing it to air dry in a laminar flow hood under sterile conditions.

Human fibroblasts (MRC-5, ATCC, Manassas, VA, USA) and human umbilical vascular endothelial cells (HUVEC, ATCC, Manassas, VA, USA) were first resuspended in serum-free supplemented Fibroblast Growth Medium (Merck, Gillingham, UK) and Endothelial Growth Medium (Merck, Gillingham, UK) and then co-cultured by adding 100,000 of each cell type per well (200,000 cells total) in a final volume of 1 mL. Cells were incubated at 37 °C in 95% humidity and 5% CO_2_ for 48 h on both uncoated and PD-Y-coated TCP to allow the formation of tissue-like structures in the former and monolayers in the control TCP.

2.2. Induction of Acute and Chronic Wound-Like Conditions

Well-defined scratch damage was generated in the central area of each co-culture by a pipette yellow tip. To simulate the acute inflammatory status typically triggered by a wound, 100,000 U937 monocyte cells (ATCC, Manassas, VA, USA) were added to each well, along with 1 mL of an equal-parts mixture of serum-free fibroblast cell culture medium (Merck, Gillingham, UK), supplemented with HUVEC medium (Merck, Gillingham, UK), and RPMI medium (Merck, Gillingham, UK). The early phases of chronic wound conditions were also generated in a separate set of co-cultures by spiking this medium with a cytokine cocktail containing 100 ng/mL of IL-6 and 135 ng/mL of TNF-α. Two control groups were also set for both uncoated and PD-Y-coated TCP, where only mechanical scratch and no inflammatory conditions were applied. All samples were incubated in humid conditions at 37 °C, 95% humidity and 5% CO_2_ for an additional 24 h.

2.3. Assessment of Key Healing Parameters

The functional differences among the three models (i.e., control, acute, and chronic) were carefully quantified by assessing four major parameters 24 h after wound induction.

2.3.1. Cell Migration

Cell migration was assessed using an optical microscope (Zeiss, Jena, Germany). Images of the wound gap were captured at ×10 and ×20 magnification 24 h after scratching. Quantitative analysis was performed by measuring at least three different areas of the scratch area, immediately and 24 h after the damage had been induced, by an image processing software (ImageJ/Fiji, Version 1.54p) measuring the distance between the two cell fronts. Data of area damage closure were expressed in microns ± standard deviation from n = 6 and statistically analyzed by two-tail ANOVA test.

2.3.2. Angiogenesis

The angiogenesis process in conditions simulating acute and chronic wound-like conditions was assessed using a range of histochemical and biochemical parameters providing both qualitative and quantitative data. Firstly, sprouting formed by HUVEC was qualitatively assessed through observation of tube network formation by optical microscopy (Zeiss, Jena, Germany) at 20× magnification. In addition, endothelial cell immunostaining was performed using antibody against CD31 expression. The samples were fixed with 3.7% v/v parafolmadehyde (ThermoFisher, East Grinstead, UK), and the antibody non-specific binding was blocked with 1% w/v bovine serum albumin (BSA, ThermoFisher, East Grinstead, UK) in phosphate-buffered saline (PBS, ThermoFisher, East Grinstead, UK), pH = 7.4. Goat Alexa Fluor 647-conjugated anti-human CD31 antibody (Abcam, Cambridge, UK) was diluted to 1:100 in 1% w/v BSA overnight, dark conditions, 4 °C. Samples were then washed three times with PBS and their nuclei counterstained with DAPI (Abcam, Cambridge, UK). Images were acquired in a z-stack mode using a Leica confocal microscope (Leica TCS SP5, Wetzlar, Germany) by both immunofluorescence and bright field settings and subsequently merged by the microscope software. Images were taken at ×10 and ×20 magnification, and the volume occupied by the formed structures is expressed as microns over the x, y, and z axes.

2.3.3. Deposition of ECM Components

The synthesis and deposition of newly synthesized ECM macromolecules was assessed qualitatively within and at the margin of the scratch area, while their quantitative measurement was performed in the co-culture supernatants. Samples were fixed with 3.7% v/v paraformaldehyde (ThermoFisher, East Grinstead, UK) and then stained with histochemical dyes for polysaccharides and collagen.

The distribution of glycosaminoglycans (GAGs) such as hyaluronic acid in the wound area was assessed using Alcian Blue staining (Sigma Aldrich, Gillingham, UK), while collagen deposition was visualized using Picrosirius Red staining (Abcam, Cambridge, UK). Both staining procedures were done following the suppliers’ instructions. Briefly, fixed cells were stained with 1% w/v Alcian Blue solution at pH 2.5 for 30 min. They were then rinsed with deionized water and dehydrated. For collagen evaluation, fixed cells were stained with 0.1% w/v Picrosirius Red solution for 60 min. They were washed in water to remove excess stain and dehydrated. Microscopy images were captured by an optical microscope at ×20 magnification (Zeiss, Jena, Germany).

To conduct a quantitative collagen assay, proteins were precipitated from the cell culture supernatant by mixing samples with cold ethanol. Then, the pellet was collected by centrifugation and resuspended in 0.1% w/v Picro-Sirius Red (Abcam, Cambridge, UK) dye solution. The mixture was then incubated for 1 h to allow the sulfonic acid groups of the dye to bind specifically to the basic groups of the collagen fibers. After incubation, the samples were centrifuged to isolate the stained collagen–dye complex. The excess dye was washed away with deionized water. The resulting red pellet was re-solubilized in 0.1 M NaOH, and the levels were spectrophotometrically measured at a wavelength of 540 nm by a microplate reader (ASYS, Eugendorf, Austria). Absorbance data were converted into concentration (μg/mL) from a standard curve ranging between 3.9 and 31.2 mg/mL of Collagen Type I (Sigma Aldrich, Gillingham, UK).

GAG content in cell culture supernatant was measured using the Alcian Blue dye-binding assay. First, samples were incubated with 0.1% w/v Alcian Blue (Sigma Aldrich, Gillingham, UK) solution, which allowed the dye to bind to sulfated GAGs. Next, centrifugation was done to separate the dye-GAG complex. The pellet was then dissolved in 4 M guanidine-HCl (Merck, Gillingham, UK). The absorbance was measured at 620 nm using a microplate reader (ASYS, Eugendorf, Austria) and the GAG concentration calculated using a hyaluronic acid (Merck, Gillingham, UK) standard curve in the concentration range of 3.5 to 62.5 mg/mL.

2.3.4. Inflammatory Cell Differentiation (Macrophage Polarization)

Immunostaining was used to analyze the differentiation of the U937 monocyte cell line in the different wound-mimicking conditions. Samples were fixed by 3.7% v/v paraformaldehyde and blocked with 1% w/v BSA in PBS as described above, and immunostained for M1 phenotype by a 1:100 dilution of rabbit anti-human induced nitric oxide synthase (iNOS) primary antibody (Abcam, Cambridge, UK) and goat anti-rabbit Alexa Fluor 488 (Abcam, Cambridge, UK) secondary antibody. Likewise, the M2 anti-inflammatory phenotype was identified by the expression of the mannose receptor detected by the 1:100 diluted rabbit anti-human Alexa Fluor 488-conjugated CD206 antibody (Abcam, Cambridge, UK). Images were taken at different magnifications and zoom using a Leica confocal microscope (Leica TCS SP5, Wetzlar, Germany).

2.4. Validation of the Model in Studies of Wound Dressing Biocompatibility

The validation of the model was pursued by analyzing the wound healing in acute and chronic conditions in the presence of two types of clinically available wound dressings, i.e., N-A Ultra (KCI, Athlone, Ireland) and Kaltostat (ConvaTec, Deeside, UK). The selection of wound dressings for model validation was based on the established effect that their biomaterial composition and engineering exert on inflammatory cells [28]. In particular, N-A is a cellulose-based biomaterial manufactured in the form of a tulle, used in clinics as a primary dressing to protect the wound from mechanical insults. It is applied to both acute and chronic wounds either on its own in cases of low exudate volume or in combination with a secondary hydrogel dressing when excess exudate needs to be absorbed. Kaltostat is an alginate-based hydrogel with high swelling capacity and relatively fast degradation rate, and it is used mainly in traumatic wounds such as burns. The substantial physicochemical differences between the two dressings were deemed an opportunity to validate the organotypic model by assessing how the presence of a foreign biomaterial in the wound bed could alter the model’s response in terms of the formation of new tissue-like structures as well as collagen and GAG deposition within the damaged area.

The wound models were induced as previously described, and either N-A Ultra or Kaltostat dressing samples (1 cm × 1 cm) were gently placed on top of the co-cultures following the scratch procedure and the addition of monocyte cell lines under conditions simulating the early phases of either acute or chronic conditions. All the healing parameters were assessed after a further 48 h of incubation as reported above, with the exception of the quantitative analysis of collagen, GAGs, and macrophage differentiation in samples treated with Kaltostat. These were omitted as both the adsorption and absorption of these biomarkers onto the dressings prevented the reliable assessment of their concentrations in the supernatants.

All experiments were performed in triplicate, and quantitative data were statistically analyzed by ANOVA two-tail test for equal variance.

3. Results

3.1. Morphological Analysis of PD-Y-Induced Dermis Organotypic Fibroblast/HUVEC Co-Cultures

The morphological analysis of the fibroblast/HUVEC co-cultures after 48 h of incubation confirmed the ability of PD-Y to induce the organization of cells into tissue-like structures and different reorganization patterns when acute and chronic wound-like conditions were applied (Figure 1A). In particular, prior to scratching, fibroblasts appeared to envelop the tubular structures formed by the HUVEC angiogenic sprouting and form compact and interconnected 3D microtissues. These structures gradually reformed 24 h after scratching in samples where an acute wound was simulated, whereas the model mimicking a chronic wound displayed frustrated angiogenesis sprouting and fibroblast reorganization. In the case of co-cultures performed on uncoated TCP, a typical monolayer developed during the first 48 h of culture and was observed to fill the gap produced by the scratching (Figure 1B).

In the organotypic co-cultures simulating both the acute and chronic wounds, closure of the scratch area was not complete, unlike in the control TCP monolayers (Figure 1A,B) and in the PD-Y organotypic groups where inflammatory cells were not added (Figure 2A,B, p < 0.001). Additionally, a noteworthy difference was identified in the gap areas’ values between the models simulating chronic and acute wounds, with delayed fibroblast migration in the chronic wound model compared with the acute wound model (p < 0.01) (Figure 2A,B, Acute Wound Model and Chronic Wound Model).

Immunostaining for CD31 expression, combined with DAPI counterstaining and analyzed either by z-stack imaging or by merging the immunofluorescence with bright field microscopy, confirmed the HUVEC composition of the tubular structures and provided quantifiable indications of the volume occupied by the vascularized, dermis-like structures, which appeared larger in the control than in the models simulating the acute wounds (Figure 3A,B, Control PD-Y). In the volume considered by the z-stack analysis, the tissue-like structure densely occupied a volume (x, y, z axes) of approximately 2 × 2 × 1.6 μm (Figure 3A, Control PD-Y). In the model simulating the onset of acute wounds, the tissue-like structures occupied a less dense volume of approximately 2 × 2 × 1.0 μm, while the expression of CD31 seemed preferentially localized at the anastomotic junctions, suggesting an infiltration of small sprouting tubes within the overlying fibroblast micro-masses rather than larger interconnecting tubules (Figure 3B, Acute model). The analysis of the endothelial cell behavior under chronic conditions clearly showed frustrated angiogenesis characterized by a relatively disorganized network of thinner and often discontinuous tubules (Figure 3B, Chronic model, green arrows).

Likewise, the deposition and organization of extracellular matrix components (i.e., collagen and GAGs) in the organotypic control were observed both intracellularly and extracellularly, with the extracellular deposition showing areas of early orientation (Figure 4, Control PD-Y, Picrosirius Red and Alcian Blue). In both the acute and chronic wound-simulating conditions, collagen staining was mainly observed in the immediate pericellular space, with no significant orientation. GAGs appeared localized intracellularly, except in areas where cells formed denser structures, in which some extracellular deposition was observed (Figure 4, Acute wound model and Chronic wound model, Picrosirius Red and Alcian Blue).

Table 1 shows the levels of collagen and GAGs released by the cells over 24 h after scratching in the organotypic culture control, where monocyte cells were not added, as well as under the two conditions simulating the acute and chronic wounds. The levels of collagen were not significantly different when the acute wound-like conditions were compared to the control scratch organotypic culture. However, both the control and the acute wound-simulating co-culture showed levels of collagen lower than those observed in the supernatants of the chronic wound-mimicking conditions.

Likewise, when compared to the control co-culture, there was an insignificant increase in the GAG levels measured in conditions simulating an acute wound, while the increase was statistically significant in chronic wound-simulating conditions.

In the models simulating the acute and chronic wound conditions, immunostaining was performed to assess macrophage polarization into either the M1 (iNOS^+^ cells) or M2 (CD206^+^ cells) phenotype (Figure 5A,B). Immunostaining of the markers showed the prevalence of iNOS^+^ cells in both the acute and chronic wound-like models, although no statistically significant difference was observed between the two conditions (Figure 5B, iNOS, Acute and Chronic Wound). Immunofluorescence also showed the tendency of iNOS^+^ cells to aggregate into clusters, particularly in the acute wound-like model (Figure 5A, iNOS, Acute and Chronic models). Only a limited presence of CD206^+^ cells was observed in the two conditions (Figure 2A,B).

3.2. Organotypic Wound Model Application to Wound Dressing Biocompatibility Studies

The validation of the model in its acute and chronic wound-like conditions was pursued by testing the effect of clinically available wound dressing products, N-A ULTRA and Kaltostat.

In the acute wound-mimicking conditions, cell migration after 48 h of treatment with N-A resulted in the complete closure of the damaged area, while the chronic wound-simulating model showed a significantly lower degree of gap closure (Figure 6A,B). The difference between gap closure in acute and chronic wound-like models was statistically significant at p < 0.001 (Figure 6B). Notably, when N-A was applied to the acute wound model, new tissue-like structures were observed aligning along the main axis of the scratch (Figure 6A,C, Acute wound model). In contrast, under chronic wound-simulating conditions, the dressing produced only partial and more randomly distributed structures with both angiogenic sprouting and fibroblast organization appearing less coordinated (Figure 6A,C, Chronic wound model). The wound dressing produced distinct effects on collagen and polysaccharide deposition under acute versus chronic wound-like conditions (Figure 6D). In the acute wound-mimicking conditions, the presence of the wound dressing appeared to restore the cells’ ability to deposit both macromolecules extracellularly (Figure 6D, Acute wound model). However, when chronic conditions were simulated (Figure 6D, Chronic wound model), the localization of these macromolecules remained largely confined within the cells and their immediate surrounding, although a modest improvement, particularly in collagen deposition in the acute wound model and GAG deposition in chronic wound-simulating conditions, was observed compared in the same condition without wound dressing application (Figure 6D, Acute wound model and Chronic wound model and Figure 4).

Analysis of macrophage polarization showed that the presence of the N-A dressing appeared to inhibit the M1 macrophage polarization (Figure 7A, left panel, Acute wound model and Chronic wound model) in favor of a shift toward M2 polarization in acute wound-simulating conditions; however, this increase in M2 cells was not statistically significant (Figure 7A, right panel, Acute wound model and Chronic wound model).

When Kaltostat was applied to the organotypic co-cultures following the scratch and monocyte seeding, a faster closure of the wound gap was observed in both acute wound conditions (Figure 8A, left microscopy images) and chronic ones (Figure 8A, right microscopy images). Indeed, measurement of the gap closure showed a statistically significant difference between the effects of the dressing in the two conditions. In the chronic wound model, gap closure improved (Figure 8B, ca 140 μm) compared with N-A treated samples (Figure 6B, 50 μm). However, the difference with the closure of the Kaltostat-treated acute wound model (Figure 8B, ca 260 μm) was statistically significant at p < 0.05 rather than p < 0.001, as observed for N-A. Likewise, collagen and polysaccharide deposition appeared to be enhanced in the acute conditions (Figure 8C).

4. Discussion

The present study successfully established, for the first time, an organotypic co-culture in vitro model that is capable of simulating the early phases of an acute and chronic wound state by using the biomimetic substrate PhenoDrive-Y. The model recapitulated many of the pathological hallmarks of chronic wounds, including impaired fibroblast migration, compromised angiogenesis, disrupted deposition of the ECM components, and inflammatory cell phenotypic changes. The alteration of these processes was shown to be partially reversed following therapeutic intervention by the application of clinically available wound dressings. Together, these findings address an important gap in modeling wounds by providing a stable, reproducible in vitro platform of enhanced translational relevance.

These results confirmed that PhenoDrive-Y supported the formation of organized tissue-like structures on a 2D plane without the need for a gel encapsulation process, whereas traditional tissue-culture plastic (TCP) lacks both structural and biochemical cues necessary to promote organotypic cultures. Especially important is the capability of PhenoDrive-Y to maintain architectural stability for >72 h. This was a significant advantage considering that many natural hydrogels, including Matrigel, degrade rapidly, making it difficult to employ them in studies exceeding three days [23,24,25]. The ability of PhenoDrive-Y to induce tissue-like structures has been ascribed to the ordered presentation of integrin bioligands, as well as to its demonstrated mesh-like topography [24]. The incubation time after scratching was deliberately limited to 24 h in the early phase of the study to enable the observation of the early phases of reorganization of the tissue-like structures while enhancing the opportunity to identify any difference occurring between the acute and chronic wound-like conditions. In the case of the model validation by testing of wound dressings, the period of observation after scratch was extended to a further 48 h to maximize any effect produced by the dressings and to simulate as closely as possible the time of application of dressings that in clinics varies from 2 to 3 days unless diabetic ulcers are treated.

In comparison with the acute wound-like model and the control, the chronic wound model showed markedly reduced fibroblast migration and closure of the gap generated by a conventional mechanical scratch. This is consistent with clinical pathology, where fibroblasts in chronic ulcers exhibit stalled motility and persist in a “non-healing” state [26]. This decreased migration corresponds with that seen by Wang et al. to show reduced migration capability under conditions of sustained pro-inflammatory cytokines [27]. These sustained pro-inflammatory conditions were simulated in this study by deliberately spiking the cells with cytokines concentration above the levels observed during the clinical studies of chronic ulcers (high rather than low nanogram scale). The observed angiogenic defects were characterized by reduced tube length and fewer branching points; this further reflects the in vivo chronic wound environment, where cytokine-induced cytotoxicity leads to endothelial dysfunction [15].

The observation of thin, fragmented collagen fibers and dysregulated hyaluronic acid distribution in ECM deposition patterns within the chronic wound model is supported by their significantly higher levels in the supernatants, reflecting the pathological features observed in non-healing human ulcers [28,29]. The initial organization of collagen bundles in the acute wound onset model is consistent with the successful transition of the cells into a proliferative phenotype and the onset of the wound-contracting phase, as observed in vivo during normal acute wounds healing [30,31]. Furthermore, the impaired collagen architecture within the chronic model was similar to the findings of Maver et al. who demonstrated ECM disorganization in hydrogels that were exposed to IL-6 and TNF-α to mimic chronic wound conditions [32]. Likewise, under conditions simulating a high inflammatory status, the cells appeared to synthesize GAGs, but these components were not released in the extracellular matrix and remained mainly localized within the cells or in their immediate surroundings.

Interestingly, macrophage polarization results showed no significant difference between the acute and chronic wound models 24 h after scratch induction and monocyte seeding, despite cytokine induction. This is perhaps reflective of the short induction period and would suggest that longer exposure may indeed be required for macrophage phenotypic divergence. This finding is supported by studies from Lacina et al. [33], which suggested that exposure longer than 48 h was required to drive a more stable M1 dominance under chronic inflammatory conditioning [19,33]. However, following therapeutic treatment by wound dressings, iNOS expression was reduced, and CD206 increased, with the strongest response observed in the acute wound model. This confirms that the system is responsive and capable of demonstrating phenotype reversal in inflammatory cells even without prior stimulation with phorbol merystate acetate (PMA) followed by IL-4 incubation. Indeed, the spiking of U937 with PMA and IL-4, which is typically employed to differentiate monocytes first into macrophages and then into the CD206-expressing M2 phenotype, was deliberately omitted in this study to ensure that any observed differentiation was driven by the microenvironment and by the contact with the wound dressings.

Treatment with N-A showed clear differences in healing potential when tested under the two conditions. The acute wound model demonstrated almost complete closure of the gap by fibroblasts within 48 h of scratch induction, along with enhanced deposition of ECM components, and evidence of macrophage repolarization. In contrast, when applied to the chronic wound model, this dressing presented only minimal improvements in the studied healing parameters, highlighting the clinical challenge of reverting long-standing chronic wound inflammation. This aligns with observations by Patel et al. and Freedman et al., who reported reduced therapeutic responsiveness in chronic ulcers [34,35]. The stimulation of all the tested parameters (i.e., angiogenesis, fibroblast organization around angiogenic sprouting and deposition of ECM components) was more evident in the case of the alginate-based dressing, Kaltostat, suggesting a scaffolding or metabolic effect of this biodegradable polysaccharide-based (i.e., alginate) dressing in tissue cells. At the same time, given the limitation of the cell counts, particularly in the case of cluster formation, the changes observed may be more cautiously interpreted as caused by differences in iNOS-labeling intensity and responsiveness to the dressings’ foreign body character rather than a fully resolved M1/M2 shift.

Few existing models have integrated fibroblasts, endothelial cells, and macrophages into one 3D structure without cell encapsulation into a gel or scaffold [36,37]. Most past models of chronic wounds depend on either the basic cell monolayer scratch assays [38] or hydrogels that degrade fast [25] or systems of a single cell type [39]. While aligning with the more advanced multicellular systems such as those described by Macedo et al. and Maver et al. [20,32,40], the wound models reported here offer improved reproducibility and the potential of dissecting specific biochemical and cellular pathways in acute and chronic wound states in their early phase. For the purpose of model standardization across laboratories, cell lines of both fibroblasts and monocytes were preferred to primary cells that are known to be prone to donors’ variabilities. While it is acknowledged that the lung fibroblasts and HUVECs used in this study do not tightly recapitulate the dermis cell populations, their widespread use and relative ease of culturing have been preferred to maximize the wide adoption of the model. Indeed, in addition to their suitability for studies of cellular and biochemical pathways of wound healing, these in vitro organotypic models also represent a meaningful step towards the standardized and reproducible preclinical screening of novel therapeutics and medical devices.

5. Conclusions

The present study describes an organotypic in vitro wound model enabling the clear-cut distinction of acute from chronic wound states in their early stage. The system recapitulated important pathological characteristics of chronic wounds, such as reduced angiogenesis, disordered extracellular matrix deposition, impaired fibroblast migration, and pathologically high levels of inflammatory signaling, while maintaining architectural integrity beyond what is possible with traditional culture platforms. The model’s responsiveness to therapeutic intervention further supports its translational value. Overall, this model overcomes major limitations of existing wound-healing systems and provides a robust, reproducible and operator-friendly platform for mechanistic research, biomaterial evaluation, and preclinical screening of potential wound therapies.