Poster BB16 - Molecular Cues of Tissue Damage Modulate Immune Response in Wound Healing

Introduction: Damage-associated molecular patterns (DAMPs) are molecules expressed on damaged tissue or released by damaged cells to facilitate an immune response in a sterile environment. DAMPs are recognized by pattern recognition receptors on macrophages and initiate the secretion of inflammatory cytokines and chemokines. DAMPs have been associated with many inflammatory diseases such as osteoarthritis, atherosclerosis, and Parkinson’s disease, and are generally regarded as one of the major underlying causes. As DAMPs are often associated with these pro-inflammatory diseases, it is easy to understand why DAMPs have yet to be explicitly leveraged in tissue engineering. The investigator’s past work (Isaac et al. Nature Communications 2024) in developing drug-loaded biomaterials for calvarial defect regeneration demonstrated that changes in the immune response at the site of injury significantly impact biomaterials-based tissue regeneration. Thus, this project aims at utilizing DAMPs as an immunomodulatory cue for wound healing. Here, we isolate tissue damage-associated cues (TDAC), derived from extracellular matrix (ECM) DAMPs, and investigate their immunomodulatory and tissue regeneration properties with the overall goal of creating an immunomodulatory tissue regenerative biomaterial (Figure 1).

Materials and

Methods: To evaluate the pro-regenerative ability of TDACs, a scratch assay was performed. TDACs were collected from damaged rat ECM (Figure 1A). Rat fibroblasts were plated on a 12-well tissue-culture plastic plate at a concentration of 200,000 cells/mL and incubated overnight to adhere. A cross-shaped scratch was made in each well using a pipette tip and imaged via light microscopy (BioTek Lionheart FX). For 16 hours, the fibroblasts were exposed to 10 %, 20%, and 30% v/v TDAC containing complete media (DMEM; 10% FBS, 1% P/S). Control groups contained complete media only. At multiple time points, the same location of the scratch was imaged (4 images per well) and analyzed for scratch area and width using ImageJ. The measurements were normalized to the initial area and gap width to determine % area and % gap closure. The media at 24 hours was collected and will be analyzed for cytokine secretion (G-CSF, GM-CSF, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, TNF alpha, and TGF-ϐ) via a multi-plex bead panel to determine the TDAC recognition receptor pathway. Fibroblasts will also be stained with a fluorescently tagged α-SMA antibody to visualize fibroblast differentiation into myofibroblast, cells primarily responsible for wound contraction.

Results, Conclusions, and Discussions: Images from the scratch assay revealed changes in the rate of wound closure as shown by the representative images in Figure 2A. When exposed to 20% v/v TDAC media, fibroblasts displayed significantly increased % area (p < 0.005) and % gap width (p < 0.01) closure when compared to the control group (Figure 2B-C). Interestingly, dose-dependent behavior was observed as fibroblasts had significantly different % area closure at 16 hours depending on the percentage of TDACs in the media. For both % area and % gap closure, 20% v/v TDACs had the highest value compared to other dosages of TDACs, with significant differences among all groups when comparing % area closure and with 10% v/v TDAC media with % gap closure. The scratch assay revealed that generally, TDACs promoted increased rates of wound closure, with a dosage of 20% v/v TDACs significantly increasing wound closure at 16 hours. This indicates that the fibroblasts recognize damaged ECM, which can influence pro-regenerative behavior. This work shows promise in the use of TDACs as an immunomodulatory therapy.

Acknowledgements (Optional): This work is supported by The University of Texas System Trauma Research and Combat Casualty Care Collaborative (175143) and the National Institutes of Health (K12 GM111726).