Novel leukocyte-depleted platelet-rich plasma-based skin equivalent as an in vitro model of chronic wounds: a preliminary study

Abstract

Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type2 Diabetes Mellitus, and long-term venous insufficiency, where poor perfusion and altered metabolism develop into a chronic inflammation that impairs wound closure. Skin equivalent organotypic cultures can be engineered in vitro to study skin biology and wound closure by modelling the specific cellular components of the skin. This study aimed to develop a novel bioactive platelet-rich plasma (PRP) leukocyte depleted scaffold to facilitate the study of common clinical skin wounds in patients with poor chronic skin perfusion and low leukocyte infiltration. A scratch assay was performed on the skin model to mimic two skin wound conditions, an untreated condition and a condition treated with recombinant tumour necrotic factor (rTNF) to imitate the stimulation of an inflammatory state. Gene expression of IL8 and TGFA was analysed in both conditions. Statistical analysis was done through ANOVA and paired student t-test. P < 0.05 was considered significant.

Results: A skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold was setup with embedded fibroblasts as dermal equivalents and seeded keratinocytes as multi-layered epidermis. Gene expression levels of IL8 and TGFA were significantly different between the control and scratched conditions (p < 0.001 and p < 0.01 respectively), as well as between the control and treated conditions (p < 0.01 and p < 0.001 respectively). The scratch assay induced IL8 upregulation after 3 h (p < 0.05) which continued to increase up to day 1 (p < 0.05). On the other hand, the administration of TNF led to the downregulation of IL8 (p < 0.01), followed by an upregulation on day 2. IL8 gene expression decreased in the scratched condition after day 1 as the natural healing process took place and was lower than in the treated condition on day 8 (p < 0.05). Both untreated and treated conditions showed a downregulation of TGFA 3 h after scratch when compared with the control condition (p < 0.01). Administration of rTNF showed significant downregulation of TGFA after 24 h when compared with the control (p < 0.01) and treated conditions (p < 0.05).

Conclusion: This study suggests that a leukocyte-depleted PRP-based skin equivalent can be a useful model for the in vitro study of chronic skin wounds related to poor skin perfusion.

Keywords: Biomaterials; Inflammation; Platelet rich plasma; Skin equivalent; Wound healing.

Fig. 1

Preparation of the leukocyte-depleted PRP-based skin equivalent. The platelet rich plasma (PRP) was mixed with 20 mM calcium chloride (CaCl2) solution and 6 × 104 cells/cm2 fibroblasts. The mixed solution was poured into a 24 mm trans-well at 37 °C in a CO2 incubator for 24 h. The CaCl2 worked as activator for the formation of autologous thrombin and allowed the PRP-based derma equivalent to solidify. At day 2 the epithelial cells were seeded on the PRP-based derma equivalent with a density of 6 × 104 cells/cm2. This skin model was cultured with an air-liquid interface system allowing the development of a multi-layered skin equivalent

Fig. 2

Morphology of the primary epithelial cells, fibroblasts, and skin equivalent. a Seeded epithelial cells showing their characteristic “pavement stone” morphology; b Fibroblasts reaching confluence 15 days after the plating of the dermal cells; c Typical air-liquid interface system composed by a PRP-leukocyte depleted scaffold in a 24 mm trans-well. The skin model was assembled through a co-culture of epithelial cells and fibroblasts-seeded in the PRP-leukocyte depleted scaffold; d Confluent epithelial cells growing in multilayers on the surface of the scaffold; e Confluent fibroblasts embedded into the scaffold; f Transversal section of the skin equivalent showing the presence of different epithelial cells organized into the different layers of the skin. Nuclei of the epithelial cells are stained in violet. The image also shows the presence of the fibroblasts (nuclei stained in violet) embedded in the PRP-leukocyte depleted scaffold stained in pink reassembling the extracellular matrix of the skin. Scale bar = 100 μm

Fig. 3

Flow cytometry to identify the epithelial cells, and mRNA analysis to verify the fibroblasts’ identity. a-f example dot plots. Flow cytometry scatter plots of the adherent epithelial cells side scatter (SSC)/ Fluorescence dot plots of each marker; epithelial cells were found positive for CD29 (a) and CD44 (b) lineages markers and also for CD90 (c), CD34 (d), CD326 (e), and CD133 (f) stemness markers; g) mRNA expression of genes involved in fibroblast characterization was determined by qPCR. Transcript levels were normalized to the ACTB reference gene using log₂ (2^-ΔCt) method. The data are presented as mean ± standard deviation (SD). The graph bar shows expression level of the genes CD90, CD73, CD105, CD45, and CD34 of cultured fibroblasts. Gene expression was confirmed by 1.5% agarose gels

Fig. 4

IL8 and TGFA Gene Expression. Bar graphs showing the significant differences between the three conditions studied: control, scratched and treated, in relation to log fold change expression levels of the genes IL8 (a) and TGFA (b). Bar graphs showing log fold change expression levels of the genes IL8 (c) and TGFA (d) of the control, scratched and treated conditions for all time points at which they were measured. The mRNA expression was determined by qPCR. Relative transcript levels were normalized to the ACTB reference gene. The expression levels of the skin models at baseline time point, before scratching, were used as a calibrator. Data presented showing the log₂ fold change. (2^−ΔΔCt) method. The data are presented as mean ± standard deviation (SD). P values were worked out through a paired student t-test. p < 0.05 was considered as statistically significant and is shown in the figure where applicable (* p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 5

Experimental wound model and the changes in the expression of the genes. Schematic representation of the effects of scratch assay and rTNF administration, on gene expression in our skin equivalent model. IL8 and TGFA expression levels were evaluated at different time points. Down arrows in red represent downregulation while up arrows in green represent upregulation