The biologica1 and mechanical functions of in-vitro generated tissues depend on the spatial arrangement of their microcostituents. Previous works, focused on controlling tissue structure by confining cells within mircogrooves or by mechanically stimulating cell-substrate constructs. However, ECM producing cells invariably migrate onto/within the substrate and the effect of migration on the dynamics of tissue assembly has not been thoroughly investigated. Recent studies started to unravel the interplay between the cytoskeleton machinery and collagen deposition. Our objective is to verify that spatial deposition of collagen is affected by ce11 migration: random migrating cells produce disordered tissues, whereas a polarized migration leads to an aligned tissue. To this aim, we used nanogrooved silicone substrates. MC3T3 cells were cultured on patterned or flat substrates (up to 4 weeks). Ce11 migration analysis was performed by time lapse microscopy of low density and high density cultures. Collagen was quantified by Sircol assay and its alignrnent was assessed by observing picrosirius stained tissues under polarized light. Microstructural analyses were performed using TEM and SEM. In the early stage of culture, MC3T3 are oriented and migrate predominantly along the nanogrooves. After one week, cells reach confluence, they are still aligned although they migrate more slowly. In contrast, no preferential direction of migratiodalignment is evident on flat substrates. At longer times, cells produce a dense multilayered collagen matrix, whose orientation is visible under polarized light. Fibres are coaligned with cells and the nanogrooves. Tissue grown on flat substrates shows local patches of cell/collagen coalignment although no macroscopic orientation is observable. These results suggest that collagen alignrnent can be controlled by guiding cells migration. The experimental setup might be a starting platform to explore different topographic patterns and to assess the optimal condition to generate tissues with predetermined orientation.

Cell-Collagen Coalignment: Controlling Tissue Architecture with Cell Migration.

VENTRE, MAURIZIO;NETTI, PAOLO ANTONIO
2010

Abstract

The biologica1 and mechanical functions of in-vitro generated tissues depend on the spatial arrangement of their microcostituents. Previous works, focused on controlling tissue structure by confining cells within mircogrooves or by mechanically stimulating cell-substrate constructs. However, ECM producing cells invariably migrate onto/within the substrate and the effect of migration on the dynamics of tissue assembly has not been thoroughly investigated. Recent studies started to unravel the interplay between the cytoskeleton machinery and collagen deposition. Our objective is to verify that spatial deposition of collagen is affected by ce11 migration: random migrating cells produce disordered tissues, whereas a polarized migration leads to an aligned tissue. To this aim, we used nanogrooved silicone substrates. MC3T3 cells were cultured on patterned or flat substrates (up to 4 weeks). Ce11 migration analysis was performed by time lapse microscopy of low density and high density cultures. Collagen was quantified by Sircol assay and its alignrnent was assessed by observing picrosirius stained tissues under polarized light. Microstructural analyses were performed using TEM and SEM. In the early stage of culture, MC3T3 are oriented and migrate predominantly along the nanogrooves. After one week, cells reach confluence, they are still aligned although they migrate more slowly. In contrast, no preferential direction of migratiodalignment is evident on flat substrates. At longer times, cells produce a dense multilayered collagen matrix, whose orientation is visible under polarized light. Fibres are coaligned with cells and the nanogrooves. Tissue grown on flat substrates shows local patches of cell/collagen coalignment although no macroscopic orientation is observable. These results suggest that collagen alignrnent can be controlled by guiding cells migration. The experimental setup might be a starting platform to explore different topographic patterns and to assess the optimal condition to generate tissues with predetermined orientation.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/389808
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