Punch-wounded, fibroblast populated collagen matrices: a novel approach for studying cytoskeletal changes in three...

Depending on growth conditions and cell type, collagen matrices populated with viable cells, i.e. commonly with fibroblasts, contract in a manner resembling wound contraction in vivo. If matrix cultures can be grown to provide the environment of contracting wounds in vitro, other conditions may be established under which fibroblasts grow reminiscent of those in normal dermis. Wounding such dermal equivalents may then initiate cells to change their phenotype in space and time in an in vivo-like environment. In turn, such a system should allow to study the underlying cytoskeletal changes at the onset of tissue repair and beyond. To test this hypothesis, we established the conditions for human skin-derived fibroblasts (KD cells) to grow within collagen matrices without contraction. We then excised from the center of such "attached, low-contracting dermal equivalents" (ALDE) "punch biopsies" with a diameter of 1 mm, and monitored the cell's shape and their microtubular networks and F-actin-containing structures over time by i) conventional fluorescence microscopy and ii) by confocal laser scanning microscopy in combination with optical sectioning and volume rendering software. Prior to wounding and in non-wounded controls up to 8 days post seeding (ps), cells predominantly exhibited an elongated, spindle-shaped morphology with distinct microtubular networks and F-actin-containing structures. Wounding induced most of the fibroblasts lining the wound edge to immediately round up. The round cells still revealed a microtubular network but only diffuse labeling for F-actin. One day post wounding (pw), these fibroblasts had resumed their spindle-shaped structure. They displayed the microtubular network and again thin F-actin-containing structures. From 2 days pw on and up to 6 days pw, the number of fibroblasts in the wound zone had increased, forming dense, multilayered patches oriented parallel to the wound surface, and the cells lining the wound margin showed the most extensive and massive F-actin-containing stress fiber-like structures. Quantification of the cell densities at the wound margin and in adjacent zones corroborated this increase, which is reminiscent of the fibroblasts migrating to the wound edge in the early phase of connective tissue repair.