Redden Robert A, Doolin Edward J
The Children's Institute for Surgical Science, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
In Vitro Cell Dev Biol Anim. 2006 Mar-Apr;42(3-4):70-4. doi: 10.1290/0509065.1.
Fibroblasts are important cellular components in wound healing, scar formation, and fibrotic disorders; and the fibroblast-populated collagen-gel (FPCG) model allows examination of fibroblast behavior in an in vitro three-dimensional environment similar to that in vivo. Contraction of free-floating FPCGs depends on an active and dynamic cytoskeleton, and the contraction dynamics are highly influenced by cell density. We investigated mechanistic differences between high- and low-cell density FPCG contraction by evaluating contraction dynamics in detail, using specific cytoskeletal disruptors. Collagen gels were seeded with human lung fibroblasts at either high (HD) or low (LD) density, and incubated with or without cytoskeletal disruptors colchicine (microtubules) or cytochalasin D (microfilaments). Gel area was measured daily. FPCG contraction curves were essentially sigmoidal, featuring an initial period of no contraction (lag phase), followed by a period of rapid contraction (log phase). Contraction curves of HD-FPCGs were distinct from those of LD-FPCGs. For example, HD-FPCGs had a negligible lag phase (compared with 3 d for LD-FPCGs) and exhibited a higher rate of log-phase contraction. Both colchicine and cytochalasin dose-dependently inhibited contraction but specifically affected different phases of contraction in HD- and LD-FPCGs; and colchicine inhibited LD-FPCGs much more than HD-FPCGs. The data indicate that LD- and HD-FPCGs contract through different primary mechanisms. Microtubules and microfilaments are both complementarily and dynamically involved in the contraction of FPCGs, and cell density influences primary cytoskeletal mechanisms. These results provide valuable information about fibroblast behavior in healing and fibrosis, and may suggest novel treatment options.
成纤维细胞是伤口愈合、瘢痕形成和纤维化疾病中的重要细胞成分；而成纤维细胞填充的胶原凝胶（FPCG）模型能够在类似于体内的体外三维环境中研究成纤维细胞的行为。自由漂浮的FPCG的收缩取决于活跃且动态的细胞骨架，并且收缩动力学受到细胞密度的高度影响。我们通过使用特定的细胞骨架破坏剂详细评估收缩动力学，研究了高细胞密度和低细胞密度FPCG收缩之间的机制差异。将人肺成纤维细胞以高（HD）或低（LD）密度接种到胶原凝胶中，并在有或没有细胞骨架破坏剂秋水仙碱（微管）或细胞松弛素D（微丝）的情况下孵育。每天测量凝胶面积。FPCG收缩曲线基本呈S形，其特征是初始无收缩期（滞后阶段），随后是快速收缩期（对数阶段）。HD - FPCG的收缩曲线与LD - FPCG的不同。例如，HD - FPCG的滞后阶段可忽略不计（与LD - FPCG的3天相比），并且对数阶段收缩率更高。秋水仙碱和细胞松弛素均以剂量依赖性方式抑制收缩，但在HD - FPCG和LD - FPCG中特异性地影响收缩的不同阶段；秋水仙碱对LD - FPCG的抑制作用比对HD - FPCG的抑制作用大得多。数据表明，LD - FPCG和HD - FPCG通过不同的主要机制收缩。微管和微丝均互补且动态地参与FPCG的收缩，并且细胞密度影响主要的细胞骨架机制。这些结果提供了有关成纤维细胞在愈合和纤维化中的行为的有价值信息，并可能提示新的治疗选择。
