Introduction: Hypertrophic scarring after burn injuries occurs in approximately 91% of all burn patients worldwide. The main pathology underlying hypertrophic scarring is fibrosis and one of the hallmarks of dermal fibrosis is the transformation of fibroblasts to alpha-smooth muscle actin (a-SMA) expressing myofibroblasts. We have developed a high-throughput screening (HTS) assay to identify novel drugs that can inhibit transformation of fibroblasts to myofibroblasts and therefore can be developed for treatment of burn scarring. Materials & Methods: Fibroblasts were derived from hypertrophic scars of burn patients. Exposure to TGF-b1 was used to induce transformation to myofibroblasts. Quantification of a-SMA and other markers was performed using immunocytochemistry (ICC), real-time qPCR (RT-qPCR) and Western blotting. The In-Cell ELISA (ICE) method was used to develop the HTS assay, where a-SMA expression and cell numbers were quantified. Results: Fibroblast identity of primary patient-derived cells was confirmed by the expression of vimentin and the absence of desmin and cytokeratin-14 expression. TGF-b1 was shown to induce myofibroblast transformation in these cells, with a 3-fold increase in expression of a-SMA. The HTS assay was able to reliably quantify TGF-b1 induced myofibroblast transformation, with early optimization of the screening assay yielding a Z factor greater than 0.5. Conclusions: The assay has been validated for HTS and will be used to screen an FDA-approved drug library of 1,900 compounds, to see if any can be repurposed as a treatment for burn scars. This is advantageous as the drugs are already deemed safe in patients. It is also likely that this approach will prevent the need for multiple surgical interventions, saving both time and money for clinicians and patients alike.
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