Full cell infiltration and thick tissue formation in vivo in tailored electrospun scaffolds

Abstract

Electrospun (ESP) scaffolds are a promising type of tissue engineering constructs for large defects with limited depth. To form new functional tissue, the scaffolds need to be infiltrated with cells, which will deposit extracellular matrix. However, due to dense fiber packing and small pores, cell and tissue infiltration of ESP scaffolds is limited. Here, we combine two established methods, increasing fiber diameter and co-spinning sacrificial fibers, to create a porous ESP scaffold that allows robust tissue infiltration. Full cell infiltration across 2 mm thick scaffolds is seen 3 weeks after subcutaneous implantation in rats. After 6 weeks, the ESP scaffolds are almost fully filled with de novo tissue. Cell infiltration and tissue formation in vivo in this thickness has not been previously achieved. In addition, we propose a novel method for in vitro cell seeding to improve cell infiltration and a model to study 3D migration through a fibrous mesh. This easy approach to facilitate cell infiltration further improves previous efforts and could greatly aid tissue engineering approaches utilizing ESP scaffolds. Statement of significanceElectrospinning creates highly porous scaffolds with nano-to micrometer sized fibers and are a promising candidate for a variety of tissue engineering applications. However, smaller fibers also create small pores which are difficult for cells to penetrate, restricting cells to the top layers of the scaffolds. Here, we have improved the cell infiltration by optimizing fiber diameter and by co-spinning a sacrificial polymer. We developed novel culture technique that can be used to improve cell seeding and to study cytokine driven 3D migration through fibrous meshes. After subcutaneous implantation, infiltration of tissue and cells was observed up to throughout up to 2 mm thick scaffolds. This depth of infiltration in vivo had not yet been reported for electrospun scaffolds. The scaffolds we present here can be used for in vitro studies of migration, and for tissue engineering in defects with a large surface area and limited depth. IntroductionElectrospun (ESP) scaffolds are highly porous and consist of nano-or micrometer sized fibers of natural or synthetic polymers, mimicking the fibrous composition of tissue extra cellular matrix (ECM) [1][2][3] . ESP scaffolds provide more mechanical support than hydrogels and are more flexible than scaffolds produced by additive manufacturing, making them interesting for tissue engineering approaches [4] . Large ESP mats are easily produced but are often limited to a thickness of several mm due to delamination and charge distribution. This makes ESP scaffolds particularly interesting for defects with a large surface area, but limited depth. This includes skin patches [5] , corneal repair [6] , cartilage regeneration [7] , vascular grafts [8] and nerve guides [9] , among others. However, due to dense fiber packing and small pores, deep cell infiltration in ESP scaffolds remains a challenge [10, 11] . To create new fully functional tissue, ESP...

Computed Tomography as a Characterization Tool for Engineered Scaffolds with Biomedical Applications

“…An amount of 0.3% PTA in 70% ethanol was used as staining and porosity, and specific surface area and percolation diameter were evaluated to establish the transport pathways through the pore space [69]. Membranes and films were also characterized through CT, revealing characteristic morphostructural features or significant differences in terms of pore size, structure thickness, or specific area between different compositions [126][127][128].…”