Department of Physiology and Biochemistry and Centre of Molecular Medicine and Biobanking, Faculty of Medicine and Surgery, University of Malta, Msida, MSD2080, Malta.
National Blood Transfusion Centre and Department of Pathology, University of Malta, Msida, MSD2080, Malta.
BMC Mol Cell Biol. 2021 May 10;22(1):28. doi: 10.1186/s12860-021-00366-6.
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.
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).
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.
慢性腿部溃疡与血红蛋白疾病、2 型糖尿病和长期静脉功能不全有关，其中灌注不良和代谢改变发展为慢性炎症，从而阻碍伤口愈合。组织工程皮肤等效物可以在体外构建，通过模拟皮肤的特定细胞成分来研究皮肤生物学和伤口闭合。本研究旨在开发一种新型生物活性富血小板血浆（PRP）白细胞耗尽支架，以促进对慢性皮肤灌注不良和白细胞浸润低的患者常见临床皮肤伤口的研究。通过划痕实验模拟两种皮肤伤口条件，即未经处理的条件和用重组肿瘤坏死因子（rTNF）处理以模拟炎症状态刺激的条件，在皮肤模型上进行划痕实验。分析两种情况下白细胞介素 8（IL8）和转化生长因子 α（TGFA）的基因表达。通过方差分析和配对学生 t 检验进行统计学分析。P<0.05 被认为具有统计学意义。
建立了一种由白细胞耗尽的 PRP 支架组成的皮肤模型，其中嵌入成纤维细胞作为真皮等效物，并接种角质形成细胞作为多层表皮。IL8 和 TGFA 的基因表达水平在对照和划痕条件之间有显著差异（p<0.001 和 p<0.01 分别），以及在对照和处理条件之间有显著差异（p<0.01 和 p<0.001 分别）。划痕实验诱导了 3 小时后 IL8 的上调（p<0.05），并持续增加至第 1 天（p<0.05）。另一方面，TNF 的给药导致 IL8 的下调（p<0.01），随后在第 2 天上调。第 1 天划痕后，IL8 基因表达在自然愈合过程中下降，第 8 天低于处理组（p<0.05）。未经处理和处理条件在划痕后 3 小时与对照条件相比均显示 TGFA 的下调（p<0.01）。与对照相比，rTNF 给药后 24 小时 TGFA 显著下调（p<0.01），与处理组相比也显著下调（p<0.05）。
本研究表明，白细胞耗尽的 PRP 基皮肤等效物可作为研究与灌注不良相关的慢性皮肤伤口的体外模型。
