Skin equivalents represent the first examples of three-dimensional organotypic cultures which are often used for in vitro research study of normal and abnormal skin biology [17, 31].
In this study, we developed a new wound model on which to observe the immune response during the wound healing process through the expression of IL8 and TGFA. The control condition was not treated, the treated condition was given an additional inflammatory stimulus through the administration of 30 ng/mL rTNF which regulates genes that code for inflammatory mediators [32], while the study condition was administered with 30 ng/mL rTNF and 6 × 104 cells/cm2 MSCs. This model is shown in Fig. 7.
Studies on monolayer cultures of keratinocytes submerged into culture media do not resemble the true nature of the physiological process of wound healing [33]. Multi-layered differentiated models are comparable to native skin and produce excellent results when analysing epithelial attachment, proliferation, differentiation, and dermal remodelling [34]. Such models support further expertise of skin biology and skin diseases, without the complexity of the intrinsic interactions found in native skin [34, 35].
One example of a skin model cultured in an air-liquid interface system is the In Vitro Reconstructed Human Epidermis [36], which does not contain fibroblasts. On the other hand, de-epidermidised dermis and collagen framework, which includes only the fibroblasts, basement membrane, and extracellular matrix (ECM) components and which are the most commonly used dermal equivalents [34] is not sufficient to reproduce a skin model. It was shown that the inclusion of fibroblasts enhances the production of extracellular matrix proteins, generating a more normal epidermal architecture [34]. Therefore, the interaction between epidermal and dermal components is needed for adequate wound healing. In another study, a skin equivalent was developed by implanting keratinocytes onto the upper surface of a collagen scaffold, occupied with fibroblasts and culture at the air-liquid interface [37].
It is known that platelet-rich plasma contain more than 300 biologically active molecules containing growth factors and pro-inflammatory and immune-modulating cytokines that can activate the platelets themselves, perpetuating the inflammatory cycle [27].
Moreover, the platelets trapped into the fibrin matrix release the growth factors slowly over a duration of seven days, in contrast with the use of exogenous thrombin, where almost all growth factors are discharged during the first hour [38].
Several studies reported positive results on the application of PRP in stimulating the wound healing process [39] and increasing keratinocyte migration [35]. Previous attempts to use platelet-derived products were tested in skin tissue engineering. One study described the use of platelet lysate, combined with chitosan and 107 hyaluronic acid dressing [40]. At the same time, others used platelet lysate in conjunction with a collagen/gelatin scaffold [41] or a collagen type I gel which was mixed with PRP [42]. These studies all showed promising outcomes, supporting the approach of the use of platelet-derived products in wound healing.
Our novel skin model consisted of a leukocyte-depleted, platelet-rich plasma scaffold, with embedded fibroblasts, as dermal equivalent and seeded keratinocytes on it as multi-layered epidermidis (Fig. 7). 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 for the mimicking of typical chronic wounds of patients with poor skin perfusion and low leukocyte infiltration. The leukocyte-depletion allowed for the evaluation of the immunomodulatory properties of the infused MSCs, which modulate the IL8 and TGFA secretion.
We subsequently used our new wound model to analyse cytokine gene expression under three conditions: control, treated, and study conditions.
In physiological wound process, the ECM components, such as fibronectin, glycosaminoglycans and collagens, regulate the dynamic and interactive process of wound healing. [43] The platelets are early modulators of the healing process [44] and the blood clot formed upon platelets activation provides a provisional “scaffolding” containing fibrin molecule and plasma fibronectin. This occurs during the first 24 hours after the injury and enables formation of a temporary matrix in the wound bed. [45] Therefore, our PRP-based scaffold as dermal equivalent reassembles the physiological scaffolding formed during the hemostatic phase and required for the normal wound process.
The initial wave of inflammatory phase is characterized by IL8 production by platelet α-granules and skin resident cells to reduce blood loss and fill the tissue gap with a blood clot rich in platelets, macrophages, leukocytes and mast cells producing/secreting cytokines and growth factors [46–49].
The inflammatory response occurs within hours of the occurrence of the damage as a localized or systemic protective response. It 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 [50]. TLRs activation in response to injury and inflammation is responsible for the upregulation of IL8 [51–53].
A significant upregulation of IL8 expression was noted three hours after the scratch injury when compared to the levels exhibited just before, thereby confirming the success of our scaffold in mimicking the wound. On the other and, 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 their TLR receptors causes downregulation of TLR2 and TLR4, most likely as a self-regulatory mechanism to prevent overactive skewing of the immune response [54]. In our model we noted a significant down regulation of IL8 following the administration of rTNF which appears to indicate the delayed the activation of the inflammatory response. The progression of IL8 in the treated group occurred in delay (i.e., at a later time point) when compared with the control group.
TLR ligation triggers the release of inflammatory mediators initiating innate immune responses mainly through the activation of macrophages, neutrophils, leucocytes, and stromal cells including MSCs, thus creating an inflammatory environment [55–56].
Neutrophils and monocytes/macrophages represent the key cells of the inflammatory phase [57] as their simultaneously release of large number of cytokines and growth factors are crucial to initiate the next phase of the healing process [58]. Neutrophils appear in the wound area a few minutes after the injury [59] and are replaced after two or three days by monocytes that undergo a transformation into macrophages [60]. Macrophages are cells of great importance for the healing process [61] as they participate in phagocytosis and are also the main source of cytokines and growth factors stimulating the proliferation of fibroblasts and collagen biosynthesis [62–63].
It was noted that a decreased influx of neutrophils in the first 4 days after the infliction of a wound has a negative impact on healing outcomes. [64]
It is well known that macrophages switch phenotypes from an M1 pro-inflammatory phenotype to an M2 pro-repair phenotype leading to the reduction of inflammatory markers and the promotion of the proliferation phase. [65]. Moreover, macrophages secrete PDGF, TGF-𝛼, and bFGF, which modulate the epithelialization, collagen accumulation, and angiogenesis. [66]. During the proliferative phase there is an increase in migration and proliferation of fibroblasts and endothelial cells as well as keratinocytes, which secrete bFGF, EGF, VEGF, bFGF, and PDGF, TGF-𝛼 and KGF. TGF-a mRNAs were isolated in both wound macrophages [67] epidermal keratinocytes at the wound edge. [68]. Based on its expression level, TGFA can be considered as a biomarker of the early phase of re-epithelialization. [69]
The results obtained with our model indicate that all three conditions studied were in an inflammatory state throughout the study as shown by the lower expression of TGFA when compared with IL8.
The absence of leucocytes, which promote the resolution of the inflammation by releasing numerous potent cytokines, probably led to a delay of the proliferative phase.
In experimental models, the stimulation of MSCs with the pro-inflammatory cytokine TNF upregulates expression of a subset of TLRs, thus increasing the sensitivity of MSCs to the inflammatory milieu [70]. We postulate that the MSC infusion could modulate the expression of IL8 and that the decrease of IL8 expression in the study condition at day 2 and especially at day 4 could indicate that the presence of MSCs inhibited the inflammatory response in contrast with an increase in the treated condition.
TLR4 receptor activation triggers the MSC1 population which exhibits a pro-inflammatory profile while activation of the TLR3 receptor activates the MSC anti-inflammatory phenotype MSC2 [14, 15, 71]. MSCs are known to display an anti-inflammatory phenotype in an inflammatory environment as characterized by increased mRNA expression of IL6 [72].
Our data suggest the possibility that the MSCs modulate the inflammatory response, switching from an immunosuppressive phenotype to a pro-inflammatory phenotype and regulating the IL8 expression. Presumably, this switch in our model occurred between day 4 and day 8, showing a substantial increase of mRNA expression in the study condition in day 8 when compared with the treated condition.
TGFA was down regulated throughout all time points in all the three conditions in our study. Interestingly, the changes in expression of TGFA had a similar pattern of to the changes in the expression of IL8 between days 2 and 8 in the study condition. We hypothesize that the modulation of IL8 could affect the expression level of TGFA.
Keratinocytes and fibroblasts could have both contributed equally to synthesize IL8.
We also suggest that the presence of a pro-inflammatory cytokine (TNF) stimulates MSCs to exert their immunomodulatory properties secreting directly IL8 or/and having a paracrine effect on IL8 and TGFA production by acting on the resident skin cells.
Further investigation is necessary to address the specific cellular source of IL8 and TGFA production and the cellular target of the MSCs paracrine action and hence to evaluate the clinical relevance of the infusion of MSCs in a scenario of a low blood supply in the site of the wound.