Relevance of the Interfacial Constraints of Fibronectin for Cell Matrix Adhesion

Protein exchange experiments, as summarised in the preceding sections, reveal the strength of protein-substrate interactions and the resulting influences on protein conformation. The obtained insights gain special importance where they provide new options for the modulation of the functionality of the immobilised proteins towards living systems. Along that line, the presented results were successfully employed to elucidate recent findings of in vitro cell adhesion experiments, which are described in detail elsewhere (Pompe et al. 2003a, 2005a). Briefly, endothelial cells were grown on the described set of hydrolysed maleic anhydride copolymer surfaces with a pre-adsorbed FN layer on top. The cell-driven reorganisation of FN into fibrillar structures was analysed at 50 min after cell seeding. The obtained fibrils, as exemplarily shown in Fig. 14, exhibit distinct differences in density and length depending on the above-described characteristics of the substrate surface. A two-dimensional autocorrelation analysis was applied to reveal periodic features in the fibrillar pattern (Pompe et al. 2004b). As shown in Fig. 15, the dependency of the FN patterns on the substrate characteristic was found, which could be correlated to the FN-substrate anchorage reflected by the time constant of FN heteroexchange experiments (taken from Table 2). The mean distance of the FN fibrils was determined to be in the range of 2.6 ± 0.7 ^m for the POMA substrate with the high adsorption strength of FN, down to 1.8 ± 0.4 ^m for the PEMA substrate with the lowest FN anchorage.

Beyond the obvious mechanistic interrelation of distinct interfacial modes of a given adhesive protein and the resulting characteristics of the cell-matrix adhesion at the artificial substrate, a most important consequence of this functional modulation of FN was observed: only the weakly v ■ -■ . M n ■.■/

Fig. 14. LSM image of rhodamine-conjugated FN reorganised by endothelial cells on a PPMA substrate. Scale bar: 20 |m

Fig. 15. FN fibrillar spacing is dependent on the time constant ta of FN heteroexchange bound FN layers on the less hydrophilic copolymer substrates were found to enable the differentiation of adherent endothelial cells into capillary networks - a process required for almost any engineered tissue (Pompe et al. 2004a).

These observations allow us to conclude that protein heteroexchange experiments are a most adequate tool for quantitatively unravelling and predicting cellular processes in contact with biomaterials.

Fig. 14. LSM image of rhodamine-conjugated FN reorganised by endothelial cells on a PPMA substrate. Scale bar: 20 |m

Fig. 15. FN fibrillar spacing is dependent on the time constant ta of FN heteroexchange

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