Controlling cell behaviour with nanopatterned substrates

The biologica1 and mechanical functions of in-vitro generated tissues largely depend on the spatial arrangement of their microcostituents. Previous works, focused on controlling tissue structure by confining cells within mirco-grooves or by mechanically stimulating cell-substrate constructs. Since cells invariably crawl on synthetic platforms, it is expected that ce11 motion affects matrix organization, but little is known on this interaction. We hypothesize that a dynamic and reciproca1 interplay between ce11 migration and tissue structure exists. In particular, spatial deposition of collagen is affected by the way cells move: randomly migrating cells produce a disordered tissue whereas a polarized migration produce an aligned tissue. To test this, we used nanograted silicone substrates. MC3T3 cells were seeded on pattemed or flat silicone substrate and kept in culture up to 4 weeks. Cell migration analysis was performed by time lapse microscopy in low density (0-24h) and high density (>lwk) conditions. Collagen was quantified by Sircol assay and its alignment was observed using polarized light on picrosirius stained tissues. Microstructural analyses were performed by means of TEM and SEM. In the early stage of culture, MC3T3 are oriented and migrate predominantly along the nanogrooves. After one week, cells reach a confluent state, they are still aligned although migrating more slowly. In contrast, no preferential direction of migrationlalignment is evident on flat substrates. At longer times, cells produce a dense multilayered structure, whose orientation is visible under polarized light. Fibres are coaligned with cells and the nanogrooves. Tissue grown on flat substrates shows local patches of celllcollagen coalignment although no macroscopic alignment is evident. These results suggest that collagen alignment can be controlled by guiding cells migration. The experimental setup might be a starting platform to explore different topographic pattems and to assess the optimal condition to generate tissues with predetermined orientations.