T8 - Comparison of Fibroblast Activity in Response to Extracellular-Matrix Motifs (mECMs) Exposure In-Vitro

Introduction: : Chronic wounds affect approximately 2.5% of the United States total population as of 2022 due to their inability to heal without intervention. As an ongoing issue, they warrant the development of innovative therapeutics for treatment. Current research explores leveraging molecules that are released from damaged tissue following injury, referred to as damage-associated molecular patterns (DAMPs) due to their ability to activate the innate immune system and initiate wound healing. This work expands on this effort by investigating the potential role extracellular matrix motifs (mECMs) may have in promoting wound healing by exploring their effect on fibroblasts in-vitro.

Materials and

Methods: : Extracellular matrix motifs (mECMs) were fabricated via fragmentation of rat tail collagen I and presented to fibroblasts either freely or bound at different dosages (10%, 20%, and 30% v/v). To evaluate the effects mECMs have on cell viability, a live/dead assay (Thermofisher, L3224) was performed on fibroblasts 24 hours after incubation. Fibroblast morphology (actin area, nuclei area, and cell elongation) were also assessed 24 hours after incubation. Proliferation was evaluated at timepoints days 1,3, and 5 following staining with ActinGreen 488 (Invitrogen, R37110) and NucBlueFixed Cell Stain (Invitrogen, R37606). Moreover, the migration of fibroblasts in response to mECM exposure was quantified based on the closure of a vertical scratch over a period of 30 hours. All analysis and quantification was performed using an Operetta CLS High-Content Analysis System(PerkinElmer, Waltham, MA) and Harmony High-Content Imaging Analysis (Perkin Elmer) software.

Results, Conclusions, and Discussions:: Following 24-hours of exposure, live/dead staining showed that mECMs did not have a negative affect on cell viability, indicating they are non-cytotoxic up to a 30% v/v dosage regardless of the exposure method, demonstrating no significant differences compared to the 0% mECM control (figures 1A and 1B). Cell nuclei were stained to evaluate proliferation and showed no statistical differences between methods of mECM exposure after 1 day, yet depicted differences on days 3 and 5 (figure 2A). The most notable difference in proliferation was seen in the 30% free group, which had the lowest number of nuclei, compared to the bound mECM groups indicating that the method of mECM exposure can influence fibroblast proliferation. Changes in cellular morphology after 24-hours of exposure depicted a decrease in nuclei area and increased cellular elongation in free mECM groups compared to bound mECM groups indicating a potential transition into a myofibroblast phenotype (figures 2B-2D). Evaluation of fibroblast migration via a scratch assay showed bound mECM groups were 90% closed after 16 hours, while free mECMs were only about 50% closed (figures 3A-3B). Overall, these results depict significant effects of mECM presentation on fibroblasts indicating a potential to optimize the usage of mECM for effective treatment for wound healing.

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