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.

Autologous Fat Plus Platelet-Rich Plasma versus Autologous Fat Alone on Sulcus Vocalis

“…Because a high concentration of growth factors could naturally activate adipose cells to grow, we could also adjust the number of growth factors according to various injured or atrophic vocal conditions. In addition to high levels of growth factors, the depletion of WBC in PRP prevents tissue inflammation because of a reduced immune response due to WBC [46]. Therefore, the mixture of autologous fat and PRP renders more prolonged fat survival and less inflammation of the vocal folds, resulting in better and more prolonged voice outcomes.…”

A comparative profile of total protein and six angiogenically-active growth factors in three platelet products

“…It is therefore unlikely and unfeasible to procure the necessary quantities for such large-scale trials. However, human in vitro models and an animal source such as porcine platelets, could be appropriate for deeper exploration [ 46 ].…”

Possible Role of Circulating Bone Marrow Mesenchymal Progenitors in Modulating Inflammation and Promoting Wound Repair

“…In this study, we isolated circulating BMMPs focused mainly on their cellular and molecular role in wound healing. Our previously published skin wound model [ 17 ] was used to observe the immune regulatory effect of the BMMPs during the inflammatory phase of wound healing. The gene expression levels of IL-8 and CD73 and of the soluble proteins IL-8, FGF-1, and SCF were measured in nine different experimental conditions at three different time points.…”

“…The skin models were digested prior to RNA extraction. According to our previous published protocol [ 17 ] RNA was extracted from 5 × 10⁶ adherent circulating BMMPs and the skin model using the Pure Link ® RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer’s protocol.…”

“…This study aimed to determine whether an inflammatory state can trigger BMMPs to acquire their immunomodulatory and tissue remodelling properties. To achieve this, we used the skin model developed in our previous study [ 17 ] to study the behaviour and the possible effect of BMMPs in a typical chronic wound in patients with low skin perfusion and hence low leukocyte infiltration. Recombinant TNFα, as pleiotropic cytokine with a pro-inflammatory effect [ 18 ], and recombinant hepatocyte growth factor (HGF), as a paracrine cellular growth factor [ 19 ], were supplemented in order to create an inflammatory and a pro-regenerative state, respectively.…”