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.

Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity.

The aim of this study was to compare the affinity values obtained for a monoclonal antibody/antigen complex using two different techniques, surface plasmon resonance (SPR) and an enzyme linked immunosorbent assay (ELISA) approach recently described by Bobrovnik S.A. and by Stevens F.J. These two techniques can be used in particular to determine the equilibrium dissociation constant, K(D), of the complex in solution or on a surface. Bobrovnik's method gives two K(D) values that differ by a factor of 100, demonstrating that two populations of complexes are present in solution. In an initial step, one protein binds relatively weakly to the other (high K(D)) and this is followed by a conformational change in the most flexible portion of the antigen, which increases the affinity (low K(D)). Only the higher of the two K(D) values can be detected when complex formation in solution is investigated using SPR, because the interaction measured concerns the fibronectin/antibody complexes of lowest affinity. In contrast, when measuring association at the sensor surface, SPR gives an average result between the two K(D) values because complexes corresponding to both affinities can form in this situation. The constants that characterise the kinetics of the fibronectin-antibody interaction obtained by SPR and ELISA are therefore different, because the methods do not allow the same phenomena to be observed. However they are consistent and complementary.