Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

Abstract

Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150 microm, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1 microm. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Göteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between cells and the fibrous scaffold indicates that these scaffolds provide a three-dimensional physical structure; a potential candidate for neural tissue engineering repair and rehabilitation.

Directed Differentation of Human Embryonic Stem Cells in Combination of Biomaterials

“…Evidence shows that of electrospun biodegradable polymers as scaffolds not only enhances the differentiation of mouse ES cells into neural lineages but also promotes and guides the neurite outgrowth [37] . Cultivation of hESC on electrospun fibrous polyurethane scaffolds also proved successful and neuronal differentiation was observed via standard immunocytochemistry [38] . Scanning electron micrographs confirmed neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold.…”

Neural ECM mimetics

“…Via electrospinning, nanofiber scaffolds that can be used for cell culturing can be fabricated. Electrospun polyurethane scaffolds with high porosity were previously shown to induce differentiation of hESCs into neurons (Carlberg et al, 2009). In another study, PC12 cells seeded on NGF encapsulated electrospun copolymer of e-caprolactone and ethyl ethylene phosphate scaffolds were observed to exhibit enhanced neurite outgrowth (Chew et al, 2005).…”

Hyaluronic acid-based scaffold for central neural tissue engineering

“…Made of nanofibrous meshes, electrospun scaffolds exhibit high surface-to-volume ratio and high porosity, and thus mimic the hierarchical structures of laminin and collagen of the ECM, which facilitates cell and axon penetration, offers guidance cues to neurite extension and enhances scaffold-tissue integration. A lot of polymers have been employed to produce electrospun fibres, including chitosan [18], poly-L-lactic acid (PLLA) [19,20], polycaprolactone (PCL) [21], polylactic-glycolic acid (PLGA) [22], polyether sulphone [23], poly(L-lactic acid)-co-poly(3-caprolactone) [24] and polyurethane [25].…”