USE OF HUMAN MESENCHYMAL STEM CELLS-DERIVED EXTRACELLULAR VESICLES AND BIOCOMPATIBLE GELLAN GUM SCAFFOLDS FOR DIABETIC FOOT ULCERS HEALING

Abstract

Diabetic foot ulcers (DFUs) are common complications of diabetes and a relevant health topic: they are characterized by a high recurrence rate and often lead to the amputation of the interested body segment. Indeed, their treatment is quite difficult because of the impaired wound healing due to the ischemic and nervous alterations of diabetic patients. The use of human mesenchymal stem cells (hMSCs)-derived extracellular vesicles (EVs), along with a biocompatible scaffold, could be part of an innovative strategy to heal DFUs, due to their anti-inflammatory and immunomodulatory properties. Moreover, EVs are not expected to induce an immunogenic response after their in situ administration. In this study the hMSCs were obtained from primary cell cultures derived from adipose tissue – from elective abdominoplasties- amniotic membranes and umbilical cords – from elective cesarean sections – of volunteer healthy patients. Normally, they would be wasted without making the most of their hMSCs content. The tissues were processed via mechanical and/or enzymatic methods, then placed in culture flasks and dishes to obtain hMSCs adherent primary cultures. At passage 3-5, the cultures were characterized via flow cytometry and their stemness potential was confirmed inducing an osteogenic and a chondrogenic differentiation. The hMSCs-derived EVs were obtained via a tangential flow filtration process, and the expression of typical surface markers (CD63 and CD81) was evaluated via flow cytometry, then their size (up to 200 nm) was assessed via Dynamic Light Scattering. As a biocompatible scaffold was considered essential to contain EVs and elicit their healing potential in the context of DFUs, a gellan gum modified with ethylenediamine (GG-EDA) scaffold was prepared and recovered via freeze-drying. The GG-EDA scaffolds were rehydrated with 100 μL of cell suspension containing human fibroblasts - cytotypes essential in wound healing processes - then a pro-inflammatory environment reproducing the one of diabetic ulcers was created treating the cells with Tumor Necrosis Factor α (TNF-α) and Interleukin-1β (IL-1β). The protective effects of EVs were tested adding 20 μg of the isolated EVs to some scaffolds with treated fibroblasts. After 48 hours, cell viability was assessed via MTS assay, demonstrating a proliferative advantage in treated fibroblasts seeded in EVs-enriched scaffolds compared to the treated fibroblasts seeded in scaffolds not enriched with EVs. Although further studies and analyses are necessary, these preliminary results suggest that hMSCs-derived EVs have anti-inflammatory properties promoting cell proliferation, thus they could be part of a strategy for DFUs treatment.