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 <0.001andp <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 3h (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 3h after scratch when compared with the control condition (p <0.01). Administration of rTNF showed significant downregulation of TGFA after 24h when compared with the control (p <0.01) and treated conditions (p <0.05).