Ultrastructural investigation on fibroblast interaction with collagen scaffold.

Collagen-based scaffolds are used as temporary or permanent coverings to help wound healing. Under natural conditions, wound healing is affected by such factors as cell types, growth factors and several components of the extracellular matrix. Due to the complexity of the cell-to-matrix interaction, many cell based mechanisms regulating wound healing in vivo are not yet properly understood. However, the whole process can be partially simulated in vitro to determine how cells interact with the collagen scaffold in relation to such features as physico-chemical properties, matrix architecture and fiber stability. Under these conditions, cell migration into the collagen matrix can be easily assessed and causally correlated with these features. In this study, we aimed at providing a structural analysis of how NIH3T3 fibroblasts migrate and proliferate in vitro when seeded on a native type-I collagen scaffold. To this end, samples were collected at regular time intervals and analyzed by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Through this experimental approach we demonstrate that collagen is gradually frayed into progressively thinner fibrils as fibroblasts migrate into the matrix, embrace the collagen fibers with long filopodia and form large intracellular vacuoles. A key role in this process is also played by microvesicles shed from the fibroblast plasma membrane and spread over long distances inside the collagen matrix. These observations indicate that a native type-I equine collagen provides favorable conditions for simulating collagen processing in vitro and eventually for unraveling the mechanisms controlling cell uptake and intracellular degradation.