Fibroblast clearance of damaged tissue following laser ablation in engineered microtissues

Abstract

Although the mechanisms underlying wound healing are largely preserved across wound types, the method of injury can affect the healing process. For example, burn wounds are more likely to undergo hypertrophic scarring than are lacerations, perhaps due to the increased underlying damage that needs to be cleared. This tissue clearance is thought to be mainly managed by immune cells, but it is unclear if fibroblasts contribute to this process. Herein, we utilize a 3D in vitro model of stromal wound healing to investigate the differences between two modes of injury: laceration and laser ablation. We demonstrate that laser ablation creates a ring of damaged tissue around the wound that is cleared by fibroblasts prior to wound closure. This process is dependent on ROCK and dynamin activity, suggesting a phagocytic or endocytic process. Transmission electron microscopy of fibroblasts that have entered the wound area reveals large intracellular vacuoles containing fibrillar extracellular matrix. These results demonstrate a new model to study matrix clearance by fibroblasts in a 3D soft tissue. Because aberrant wound healing is thought to be caused by an imbalance between matrix degradation and production, this model, which captures both aspects, will be a valuable addition to the study of wound healing.

FIG. 1.

Tissue injury by transection and laser ablation in stromal microtissues. (a) Two modes of injury are used to generate full-thickness gaps in microtissues. Phase contrast images show tissues immediately following injury. Scale bars: 100 μm. (Left) A diamond microdissection knife was used to manually create gaps in tissues. (Right) An Nd:YAG laser pulse, at 532 or 1064 nm, focused through a 10× objective, ablates a gap in the tissues. (b) Quantification of initial gap area following injury. Laser gap sizes are shown for distinct energy levels. (c) Epifluorescence images showing Hoechst stain for all cell nuclei (blue) and ethidium homodimer-1 stain for nuclei of dead cells (green/white). Scale bars: 100 μm. (d) Quantification of percentage of tissue area covered by dead cells [from (c), white area/blue area]. Data in panels (b) and (d) were compared to the knife injury condition using the non-parametric steel test with control. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns: not significant.

FIG. 2.

Closure of injured microtissues. (a) Time-lapse images are shown at 0 , 4, and 16 h for each injury mode. The white dotted line indicates gap edges. Scale bars: 100 μm. (b) Gap area as a function of time for each injury mode. Mean and standard deviation for n = 4 tissues per mode with comparable starting areas. (c) Closure rate for the initial phase during the first 4 h of the time-lapse (left) and between 6 and 16 h (right). Data are compared to knife condition using Dunnett's test with control. *** p < 0.001 and ns: not significant. Multimedia view: https://doi.org/10.1063/5.0133478.1

FIG. 3.

Fiber disruption and breakage following ablation. (a) Hoechst dye (blue) and reflected 488 nm illumination (green). (Left) Max projection of tissue z-stacks. Scale bars: 100 μm. White box correlates with region shown on right. (Right) Single z-slices of 75× images. Scale bars: 10 μm. (b) Histogram in polar coordinates showing frequency (r-axis) of net fiber orientations ( $\theta$-axis) of 10 pixel2 subregions for 25 μm of tissue adjacent to the gap long edge. Histograms are significantly different according to Kolmogorov–Smirnov test, p < 0.001. (c) Max z-projections of time-lapse reflection microscopy. Yellow arrows show compacted tissue at gap edge at the conclusion of the clearance phase. Scale bars: 200 μm. (c′)−c‴) Enlargement of area inside white box from (c), laser injured tissue, showing fiber breakage at 3 h (c′), 4 h (c″), and 5 h (c‴). White arrows indicate breakage site. (d) Opening rate, normalized to the mean control (DMSO) rate within each biological replicate, during the first 4 h following ablation. Non-parametric steel test with control. *** p < 0.001. Multimedia view: https://doi.org/10.1063/5.0133478.2

FIG. 4.

Transmission electron microscopy suggests ECM fiber endocytosis following ablation. (a) Uninjured microtissue. (b) Schematic of where the field of view was located in the microtissues for (c)−(e). (c) Fibroblasts at gap edge fixed immediately after ablation. Red arrows indicate dying cells, characterized by reduction in cell size, fragmented nuclei, many vacuoles, and blebbing. (d) Cells at gap edge 4 h after injury. Yellow arrows indicate vacuoles containing ECM fibers. (e) Cells at gap edge 20 h after ablation. Red arrows indicate non-viable cells. The cell on the left has an enlarged nucleus and many mitochondria. (a)−(e) Scale bars: 4 μm. (F) Quantification of number of vacuoles containing distinct fibrillar structures normalized to cellular area for uninjured tissue and tissue 0, 4, or 20 h after ablation. n = 10 fields of view. (c′) Magnified view of the green box from (c), immediately following ablation. An oncotic or necrotic cell, characterized by reduction in cell size, chromatin condensation at nucleus edge, and the formation of many cytoplasmic vacuoles. (d′) Magnified view of the green box from D, 4 h after ablation. An intracellular vacuole containing fibrillar structures matching the distinct appearance of the extracellular matrix. (e′) Magnified view of the green box from (e), 20 h after ablation. Blue arrowheads indicate extracellular matrix fibers that appear to be emanating from the cell. (c′')–(e′) Scale bars: 1 μm.