Department of Ophthalmology, UT Southwestern Medical Center, Dallas, TX, USA.
Biomedical Engineering Graduate Program, UT Southwestern Medical Center, Dallas, TX, USA.
Sci Rep. 2018 Aug 22;8(1):12580. doi: 10.1038/s41598-018-30372-2.
This study utilizes high resolution multi-dimensional imaging to identify temporal and spatial changes in cell/extracellular matrix (ECM) patterning mediating cell migration, fibrosis, remodeling and regeneration during wound healing. Photorefractive keratectomy (PRK) was performed on rabbits. In some cases, 5([4,6-dichlorotriazin-2yl]-amino)fluorescein (DTAF) was applied immediately after surgery to differentiate native vs. cell-secreted collagen. Corneas were assessed 3-180 days postoperatively using in vivo confocal microscopy, and cell/ECM patterning was evaluated in situ using multiphoton and second harmonic generation (SHG) imaging. 7 days post-PRK, migrating fibroblasts below the ablation site were co-aligned with the stromal lamellae. At day 21, randomly patterned myofibroblasts developed on top of the ablation site; whereas cells underneath were elongated, co-aligned with collagen, and lacked stress fibers. Over time, fibrotic tissue was remodeled into more transparent stromal lamellae. By day 180, stromal thickness was almost completely restored. Stromal regrowth occurred primarily below the ablation interface, and was characterized by co-localization of gaps in DTAF labeling with elongated cells and SHG collagen signaling. Punctate F-actin labeling was detected along cells co-aligned with DTAF and non-DTAF labeled collagen, suggesting cell-ECM interactions. Overall, collagen lamellae appear to provide a template for fibroblast patterning during wound healing that mediates stromal repopulation, regeneration and remodeling.
本研究利用高分辨率多维成像技术，识别在伤口愈合过程中细胞/细胞外基质（ECM）模式的时空变化，这些变化介导细胞迁移、纤维化、重塑和再生。对兔子进行光折射性角膜切削术（PRK）。在某些情况下，手术后立即应用 5（[4,6-二氯三嗪-2-基]-氨基）荧光素（DTAF）来区分天然与细胞分泌的胶原。术后 3-180 天，通过活体共聚焦显微镜评估角膜，并通过多光子和二次谐波产生（SHG）成像原位评估细胞/ECM 模式。PRK 后 7 天，在消融部位下方迁移的成纤维细胞与基质板层共排列。第 21 天，消融部位顶部出现随机模式化的肌成纤维细胞；而下方的细胞伸长，与胶原共排列，并且缺乏应力纤维。随着时间的推移，纤维组织被重塑为更透明的基质板层。第 180 天，基质厚度几乎完全恢复。基质再生主要发生在消融界面下方，其特征是 DTAF 标记的间隙与伸长细胞和 SHG 胶原信号的共定位。沿与 DTAF 和非 DTAF 标记胶原共排列的细胞检测到点状 F-肌动蛋白标记，表明细胞-ECM 相互作用。总体而言，胶原板层似乎为伤口愈合过程中成纤维细胞模式提供了一个模板，介导基质再填充、再生和重塑。
