Formation of low-density electrospun fibrous network integrated mesenchymal stem cell sheet

Cell sheets combined with electrospun fibrous mats represent an attractive approach for the repair and regeneration of injured tissues. However, the conventional dense electrospun mats as supportive substrates in forming "cell sheet on fiber mat" complexes suffer from problems of limiting the cellular function and eliciting a host response upon implantation. To give full play to the role of electrospun biomimicking fibers in forming quality cell sheets, this study proposed to develop a cell-fiber integrated sheet (CFIS) featuring a spatially homogeneous distribution of cells within the fiber structure by using a low-density fibrous network for cell sheet formation. A low-density electrospun polycaprolactone (PCL) fibrous network at a density of 103.8 ± 16.3 μg cm^-2 was produced by controlling the fiber deposition for a short period of 1 min and subsequently transferred onto polydimethylsiloxane rings for facilitating cell sheet formation, in which rat bone marrow-derived mesenchymal cells were used. Using a dense electrospun PCL fibrous mat (481.5 ± 7.5 μg cm^-2) as the control, it was found that cells on the low-density fibrous network (L-G) exhibited improved capacities in spreading, proliferation, stemness maintenance and matrix-remodeling during the process of CFIS formation. Structurally, the CFIS constructs revealed strong integration between the cells and the fibrous network, thus providing excellent cohesion and physical integrity to enable strengthening of the formed cell sheet. By contrast, the cell sheet formed on the dense fibrous mat (D-G) showed a two-layer (biphasic) structure due to the limitation of cellular invasion. Moreover, such engineered CFIS was identified with enhanced immunomodulatory effects by promoting LPS-stimulated macrophages towards an M2 phenotype in vitro. Our results suggest that the CFIS may be used as a native tissue equivalent "cell sheet" for improving the efficacy of the tissue engineering approach for the repair and regeneration of impaired tissues.