Introduction Extracellular Microvesicles (EMVs) can carry genetic messages and biologically active proteins throughout tissues and the body. Because of their transport capabilities, EMVs play an important role both diseased and healthy conditions. For example, EMVs play an important regenerative role in many damaged tissues. In the current studies, we examine the role of EMVs in epithelial wound healing. The potential use of EMVs as drug delivery vehicles has gained considerable scientific interest because they can be delivered in circulation, can be targeted to specific areas/cells, and can pass natural barriers. In the current work, we investigate the potential of EMVs or EMVs loaded with growth factors as a tool to enhance cell migration in order to accelerate epithelial wound healing.
Material and Methods Spontaneously immortalized skin keratinocyte and macrophage cells were stressed for 48 h by serum free media to enhance the release of the EMVs from keratinocytes (KMVs) and macrophages (MMV). The EMVs from both cell lines were isolated and collected using a centrifugation process. Specifically, the collected serum free media were centrifuged at 4 °C (500 × g for 10 minutes followed by 2,000 × g for 20 minute). The supernatant was then centrifuged at 24,000 × g for 2 hours to isolate EMVs. EMVs were “loaded” with growth factors by incubating them for 1.5 h at room temperature with PDGF, TGF-ß, VEGF, and FGF (25ng/ml per each). These “loaded” EMVs were then ultra-centrifuged at 176,000 x g for 3 h to re-pellet the loaded microveiscles derived from keratinocytes KMVs (LKMVs) or macrophages MMVs (LMMVs).
In order to evaluate the role of microvesicles on cutaneous wound healing, we chose the in vitro wound scratch assay to evaluate the cell migration rate and the wound healing percent after adding of KMVs, LKMVs, MMVs, and LMMVs separately to Epidermal keratinocytes culture. Epidermal keratinocytes were plated into 6 well plates, and wound scratch was made using 10 µl pipette tip. The model was visualized by 10 x magnification power of EVOS XL Core Cell Imaging System and analyzed using Mat lab software to measure wound area.
MTT assay was used to evaluate the proliferative effect of KMVs, LKMVs, MMVs, and LMMVs on Epidermal keratinocytes. The loading was confirmed by using BioPlex Pro cytokine assays.
Results after 72 h, the wound area in the EMVs (KMVs & MMVs) and LEMVs (LKMVs & LMMVs) treated groups showed a significant decrease in wound area and a remarkable ability to repair the wound area as compared with the control group (P < 0.0001). The percent of wound healing was almost three times more in KMVs and MMVs treated groups (57.85 % ±3.13, 69.84 % ± 4.87, respectively), and four times in LKMVs and LMMVs treated groups (80.10% ±3.50, 90.87% ±2.00, respectively) when compared to the control groups (21.74 % ± 2.389) (P < 0.0001). Furthermore, the migration rate in the presence of KMVs and MMVs (0.008810 ± 0. 0006856, 0.01085 ±0.0007964 mm2/h, respectively) and LKMVs and LMMVs (0.01470 ± 0.0009428, 0.01767 ±0.001163 mm2/h, respectively) were enhanced when compared to the control group (0. 003820 ± 0. 0003760 mm2/h).
Conclusion EMVs and Loaded EMVs have a potential regenerative effect in wound healing, which promotes and enhances cell migration and proliferation, resulting in accelerated wound closure. Based on these finding, we suggest that EMVs are a novel and promising therapeutic tool for epithelial wound healing.