In this study we developed a novel skin wound model in order to observe the immune response during the wound healing process, by quantifying the gene expression of IL8 and TGFA and measurable protein secretion. A control skin model consisting of a wounded condition without treatment was compared with a wounded (scratched) skin model with treatment. The treatment included the administration of 30 ng/mL rTNF which regulates genes that encode for inflammatory mediators [25]. This skin model is shown in Fig. 5.
Studies on monolayer cultures of keratinocytes integrated into culture media do not resemble the true nature of the physiological process of wound healing [26]. Multi-layered differentiated models are comparable to native skin and produce excellent results when analysing epithelial attachment, proliferation, differentiation, and dermal remodelling [27].
Our novel skin model consisted of a leukocyte-depleted, platelet-rich plasma scaffold, with embedded fibroblasts, as dermal equivalent and seeded keratinocytes as multi-layered epidermis. Calcium chloride was used as an activator to initiate the formation of autologous thrombin from prothrombin, forming a fibrin clot that provided a surface for keratinocyte seeding and enabled the skin cells to mature into stratum corner and basal, spinous and granular layers. The lack of leukocytes allowed the mimicking of typical chronic wounds of patients with poor skin perfusion and low leukocyte infiltration. We subsequently used our new wound model to analyze cytokine gene expression under two conditions: scratched, treated.
During the physiological wound process, platelets are early modulators of the healing process and the blood clot formed upon platelet activation provides a provisional “scaffold” containing fibrin molecule and plasma fibronectin. This occurs during the first 24 h after the injury and enables formation of a temporary matrix in the wound bed [28]. Therefore, our PRP-based scaffold as dermal equivalent resembles the physiological scaffolding formed during the haemostatic phase and required for the normal wound process.
The inflammatory response occurs within hours of damage at the affected area, and is a localized or systemic protective response. The response is activated by molecules expressed by pathogens or associated with tissue injury and are recognized by Toll-like receptors (TLRs) present on skin resident cells [29]. TLR activation in response to injury and inflammation is responsible for upregulation of IL8 [30].
A significant upregulation of IL8 expression was noted 3 h after the scratch injury when compared to the levels exhibited just before injury, thereby confirming the success of our scaffold in mimicking the wound response. On the other hand, the scratch injury exhibited a down regulatory effect on the expression of TGFA.
In addition to the induction of inflammation by chemokines, other molecules such as TNF promote the inflammatory response following wounding. It has been shown that the prolonged stimulation of TLR receptors causes downregulation of TLR2 and TLR4, which may act as a self-regulatory mechanism to prevent an overactive immune response [31] In our model we noted a significant downregulation of IL8 following the administration of rTNF, which appears to indicate the delayed activation of the inflammatory response. An increase in IL8 in the treated group occurred at a later time point compared with the scratched group.
TLR ligation triggers the innate immune responses mainly through the activation of macrophages and neutrophils, which release a large number of cytokines and growth factors stimulating the proliferation of fibroblasts and collagen biosynthesis [32]. It is well known that macrophages alter phenotypes from an M1 pro-inflammatory phenotype to an M2 pro-repair phenotype, leading to a reduction in inflammatory markers and a promotion of a proliferation phase, secreting PDGF, TGF-α, and bFGF [33].
In the clinical setting, the presence of a chronic venous insufficiency ulcer (CVIU) in a patient may result in an impaired and difficult wound healing process due to the persistence of a chronic inflammatory state, combined with a relative lack of angiogenesis. The absence of the progression from a chronic, pro-inflammatory state, with high levels of TNF-a, IL-8, RANTES, and MIP-1p, to an anti-inflammatory state, with increased cytokines released by recruiting mononuclear phagocytes, will delay the resolution of the inflammatory phase. A study conducted on patients with CVIU, specifically on tissue and fluid from the wound area, suggest that pro-inflammatory, anti-inflammatory, and macrophage-related cytokines may be important in the pathogenesis of such disease. Interestingly, in the untreated CVIU only IL-8 was present in levels higher than in normal skin [34]. This clinical observation could explain the high levels of IL8 in our wound model when compared with the control condition.
In a study by Moore et al., CVLUs show that macrophage activation is suppressed, leading to an impaired inflammatory response and reduced levels of cytokines and growth factors, which are fundamental for the recruitment of the resident skin cells [35]. Moreover, macrophages secrete PDGF, TGF-α, and bFGF, which modulate the epithelialization, collagen accumulation, and angiogenesis [36]. It was demonstrated that TGF-alpha secretion is regulated in response to the inflammatory cytokines [37, 38].
TGF-a mRNAs were isolated in both wound macrophages and epidermal keratinocytes at the wound edge [39]. Based on its gene expression level, TGFA can be considered as a biomarker of the early phase of re-epithelialization [40]. During the proliferative phase of wound healing, there is an increase in the migration and proliferation of fibroblasts and endothelial cells, as well as keratinocytes which secrete bFGF, EGF, VEGF, bFGF, and PDGF, TGF-α and KGF.
Conditions including CVLUs, venous hypertension, and pressure ulcers, are characterized by a reduced proliferation rate and a migration of resident fibroblasts, in comparison with patient-matched normal skin fibroblasts [41,42,43,44].
Cellular elements, growth factors and cytokines simultaneously play an important role in different phases of the healing process. Altering their production or any changes in their levels could account for the impaired healing observed in chronic wounds. In diabetic ulcers, the lack of new blood vessel formation results in poor afflux of inflammatory cells to release cytokines and growth factors [45].
As in the clinical setting of the CVLU, the absence and hence non activation of the macrophages could be explaining the deregulation of TGFA in our model. The results obtained with our model indicate that the scratch assay in both the untreated and treated conditions studied induced an inflammatory state, as shown by the lower gene expression of TGFA when compared with IL8. The absence of leukocytes, which promote the resolution of inflammation by releasing numerous potent cytokines, suggests a delay of the initiation of the proliferative phase. TGFA was downregulated when measured al all time points after scratching in both the scratched and treated conditions.
From this study we can suggest that the presence of a pro-inflammatory cytokine (TNF) regulate IL8 and TGFA production, by acting on the resident skin cells. The wound model indicated accurately how the resident skin cells induce IL8 and downregulate TGFA, in response to damage in the physiological and inflammatory state and in the absence of leucocytes afflux.
Further studies are required to investigate the functionality of our skin model scaffold in producing anti-inflammatory cytokines such as IL10, IL4, IFN-alpha, TGF-beta, following the administration of a scratch which mimics a skin wound to induce an inflammatory state. This will provide a better understanding of the skin resident cell response. Additionally, the functionality of our scaffold could be tested for new tissue growth under physiological and pathological wound conditions in view of its possible application in regenerative medicine.