The scratch assay: A suitable in vitro tool for studying wound healing effects
Wounds are physical injuries that result in an opening or break of the skin. Wound healing is a complex process requiring the collaborative efforts of many different tissues and cell lineages and involves proliferation, migration, matrix synthesis, and contraction. We here report the establishment of a scratch assay allowing first insights into the wound healing properties of possible therapeutic remedies including natural compounds and plant extracts. Using Swiss 3T3 albino mouse fibroblasts in monolayers the scratch assay can be used as a model imitating the migration to and proliferation within the wound site. Wound closure is simulated by mechanically creating defects in cell monolayers stratching a line through the layer. The cell-free area is then re-populated by fibroblasts through the combined action of migration and proliferation. Quantifying is done by inspecting the open gap microscopically and analysing the cells which move in and fill the damaged area after staining with 4',6-diamino-2-phenylindole (DAPI) using CellC software.
We proved the efficacy of the assay by studying preparations from several Brazilian (Brugmansia suaveolens, Eupatorium laevigatum, Galinsoga paviflora, Iresine herbstii, Kalanchoe tubiflora, Pluchea sagitalis, Piper regnelii, Schinus molle, Sedum dendroideum, Waltheria douradinha, Xanthium cavallinesii) and European traditional medicinal plants (Calendula officinalis, Hypericum perforatum) known for their wound healing effects. The platelet derived growth factor serves as a positive control. The assay turned out to be robust and to give reproducible results.
Acknowledgement: Financial support from the government Baden-Württemberg (Zukunftsoffensive IV) is gratefully acknowledged. We thank Prof. D. Meyer and T. Wilmes, for helpful advises and Dr. J. Orth, all Institute of Experimental Pharmacology and Toxicology, for providing us with the cell line.