Introduction:: Dynamic cellular behaviors, such as motion and force, are critical phenotypes observed in various pathophysiological events. The collective migration of epithelial cells drives wound closure, which is primarily induced by crawling forces of forefront cells and purse-string contractions along the boundary. In large wounds, two distinct closing modes can occur simultaneously, resulting in "fingering instability" patterns that exhibit crawling force at the fingertips and contraction force along the concave arc between adjacent fingers, known as a valley. The rate of boundary progression is affected by boundary curvature [1], so the finger-valley shape structures at the boundary play a crucial role in determining the collective migration of the cell layer. Recent studies indicate a significant correlation between intercellular force and fingering frequency, indicating possible physical regulation of fingering extrusion from cellular dynamics in the cell layer [2]. Based on these observations, we hypothesize that the fingering patterns and cell migration have feedback during the wound-closing event. Thus, this study aims to elucidate this feedback by quantifying the dynamical traits of cells, such as traction force, cell migration, and density changes.
Materials and Methods:: For our epithelial tissue model, we utilized the Madin-Darby Canine Kidney (MDCK) cell line, a normal kidney epithelial cell line commonly employed in wound healing studies. We designed silicon wafers with engravings of various geometries to create wounds with specific curvatures, and we utilized polydimethylsiloxane (PDMS) as a soft substrate for the wound stamp. We carefully positioned the stamps onto the culture dish containing cell dispersion, allowing the cells to form epithelial layers. We then removed the stamps with care to ensure the wound shapes were undamaged.
Results, Conclusions, and Discussions:: We observed that larger wounds often exhibit rough interfaces due to cells protruding from the wound boundary, resulting in the formation of periodic waves and extruding fingers (Figure 1). Our research suggests that the frequency of fingering waves positively impacts the rate of wound closure, which is supported by our experimental demonstration of a correlation between fingering frequency and overall closure rate. We also identified cellular density and initial boundary curvature as regulating factors for fingering extrusions. Experimental results confirm that cellular density plays an upstream role in finger generation, while the initial concave boundary suppresses the fingering frequency (Figure 2). Interestingly, the fingering frequencies were regulated to achieve a certain valley curvature value independent of the initial curvature of the wound, resulting in a consistent closing speed. The relative positions of cells determine the suppression of fingering extrusion, reflecting the physical potential of each constituent cell. In conclusion, this study provides important insights into the cellular mechanisms that drive the formation of fingering structures and may inform new wound-healing strategies. It also proposes a model predicting the positive correlation between fingering frequency and the overall closing speed of the boundary. The study discovered an inverse relationship between fingering frequency and boundary curvatures, implying that the closure rate is "self-controlled" and independent of initial wound periphery curvatures.
Acknowledgements (Optional): : This research was supported by National Research Funding granted by the Korean Government (NRF-2017R1A2B2007673, NRF-2020M3A9E4039658) and by the KAIST (Basic Science Research Program for faculty members).
References (Optional): : [1] Ravasio, A. et al. Gap geometry dictates epithelial closure efficiency. Nature Communications6, 7683, doi:10.1038/ncomms8683 (2015).
[2] Vishwakarma, M. et al. Mechanical interactions among followers determine the emergence of leaders in migrating epithelial cell collectives. Nature Communications 9, 3469, doi:10.1038/s41467-018-05927-6 (2018).
