Abstract
BackgroundChronic 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.ResultsA 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).ConclusionThis 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.
Highlights
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
This study suggests that a leukocyte-depleted platelet-rich plasma (PRP)-based skin equivalent can be a useful model for the in vitro study of chronic skin wounds related to poor skin perfusion
Chronic leg ulceration has been seen in Type 2 Diabetes Mellitus (T2DM) and in long-term venous insufficiency where poor perfusion and altered metabolism set up a chronic inflammation that impairs repair and wound closure [6, 7]
Summary
Chronic leg ulcerations are associated with Haemoglobin disorders, Type Diabetes Mellitus, and long-term venous insufficiency, where poor perfusion and altered metabolism develop into a chronic inflammation that impairs wound closure. The incidence of frequent leg ulcers increases with age and is common in age, leading to a negative impact on the patient’s quality of life and a considerable cost for the health care service [1] Haemolytic disorders such as Sickle Cell Disease and β-thalassaemia are seldomly diagnosed in Malta and other Mediterranean countries due to a vast occurrence of β globin gene mutations [2]. Chronic leg ulceration has been seen in Type 2 Diabetes Mellitus (T2DM) and in long-term venous insufficiency where poor perfusion and altered metabolism set up a chronic inflammation that impairs repair and wound closure [6, 7]. The epithelial cells and the fibroblasts of the skin equivalent are capable of secreting cytokines, chemokines, and growth factors that mimic wound healing and favour skin regeneration, to provide a protective layer over the wound [9, 10]
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