Author(s)
Joanna Kam, MD
Edward Walton, MD
Xiang Guo Dai, PhD
Kevin R. Bobbitt, PhD
Lamont R. Jones, MD PhD
Affiliation(s)
Henry Ford Health System;
Abstract:
Educational Objective: At the conclusion of this presentation, the participants should be able to discuss how keloid derived exosomes impact normal fibroblast migration in vitro. Objectives: Keloids are fibroproliferative tumors that can develop during wound healing. Exosomes are extracellular vesicles whose cargo, mainly microRNA, promote cell to cell signaling during tumorigenesis and wound healing. The role of exosomes in keloid pathogenesis is not well described. The objective of this study is to describe the impact of keloid derived exosomes on normal fibroblasts using a scratch assay. Study Design: Prospective controlled in vitro study. Methods: Keloid tissue samples from the head and neck were excised and exosomes were extracted using ultracentrifugation. Exosomes were confirmed by transmission electron microscopy, then quantified using the Pierce BCA protein assay kit and Western Blotting for exosome markers (Alix and CD63). Cultures of a normal human dermal fibroblast cell line were treated with exosomes (20 µg/mL). PBS and media without exosomes were included as negative controls. Culture slides were seeded with 25,000 fibroblasts and incubated to form monolayers. Scratches were made in each monolayer using 200 µl pipette tips. Slides were incubated and representative light microscopy images were taken at 12, 24 and 48 hours using 40x magnification. Gap distances were measured using ImageJ software. Measurements were performed in sextuplicate. At 48 hours the slides were fixed and stained for Ki67 to differentiate between proliferation and migration. Results: The distances between the migrating cells was measured and subtracted from the measured distances between the edges of the scratches, to give the migrated distance for each sample. After 48 hours, the average migrated distance for the PBS, media and exosome treated groups were 0mm, 0mm and 137µm respectively. Ki67 staining suggests decreased proliferation in the exosome treated groups compared to the negative control groups. Conclusions: Increased migration distances and decreased proliferation rates were seen for normal dermal fibroblasts treated with 20 µg/mL of keloid tissue derived exosomes, compared to negative controls. This suggests that keloid tissue derived exosomes promote normal fibroblast migration.