Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials.

The aim of this work was to study, in vitro, cell colonization of two biomaterials currently used for bone and cartilage repair, this step being important to understand the function of engineered tissues. Current methods that use histological approaches are not always suited to tissue-engineering analysis. We, therefore, set up a protocol to assess cell distribution, utilizing noninvasive confocal microscopy and fluorescent labels with a far red emission wavelength to optimize scaffold transparency and minimize light scattering. Hard (ceramic substitute) and soft (collagen sponge) biomaterials were seeded respectively, on one side of the scaffold, with human fibroblasts and bovine chondrocytes labelled with carbocyanine dyes (DiD and DiR). The mean penetration depth for DiR labelled fibroblasts and chondrocytes in the two scaffolds, around 270 m, was greater than for DiD (136-218 microm) labelled cells. These depths were independent of cell origin but were influenced by the nature of the scaffolds. Collagen sponge is transparent in contrast to ceramic substitutes where measurements could only be made in opened macropores. Besides the limits of the equipment, the limits of the supports were diffusion for collagen sponges and transmission for ceramic substitutes. Confocal microscopy techniques could thus be used to address the question of cell colonization of porous biomaterials in a noninvasive manner.