Confocal laser-scanning microscopy for determining the structure of and keratinocyte infiltration through collagen sponges

The development of artificial skin substitutes based on cultured cells and biomaterials such as collagen requires an understanding of cellular interactions with the substrate. In this study, human keratinocytes were cultured on the surface of collagen sponges, and confocal laser-scanning microscopy (CLSM) was used to assess both the microstructure of the sponge, and the cell morphology and distribution throughout the sponge. It was found that the pore size increased with increasing depth into the sponge. Both pore size and fiber thickness increased during incubation for up to 10 days at 37 degrees C in culture medium in the absence of cells. This latter effect was not observed when the sponges were incubated in distilled water. Keratinocytes penetrated into the sponge even after only 3 days in culture. By 10 days in culture, the cells had penetrated to the maximum depth that could be examined (120 mu m from the sponge surface). In the presence of cells, the inner structure of the collagen sponge had altered after 10 days in culture, with the collagen fibers becoming thicker, and pore geometry less regular. The mechanism responsible for this is unknown at present. Although the presence of the keratinocytes increases distortion of the sponge structure, factors from the medium itself also contribute to this effect. CLSM is a powerful tool for assessing cellular interactions with bioimplants, providing both qualitative and quantitative information. It offers many advantages over scanning electron microscopy (SEM) and histological techniques. CLSM minimizes the time-consuming, extensive preparation of samples required with the latter two methods, and allows noninvasive serial optical sectioning of intact samples. (C) 1996 John Wiley & Sons, Inc.