Removal of Sacrificial Fibers Enhances Long Term Cell and Matrix Distribution in Aligned Nanofibrous Scaffolds

Given their ability to dictate initial cell alignment and subsequent matrix organization, aligned electrospun scaffolds are a fitting means for engineering fiber-reinforced, anisotropic tissues such as tendon, ligament, the knee meniscus, and the annulus fibrosus [1–3]. However, one commonly observed limitation of such scaffolds is the relatively slow infiltration rates of surface-seeded cells, where the central thicknesses of constructs cultured for 10 weeks remain devoid of cells [2]. This limitation arises from the tight packing of fibers which yields small pore sizes, thereby hampering cell migration. Towards accelerating cell ingress, we have recently reported on two-polymer composite scaffolds containing both slow eroding poly(ε-caprolactone) (PCL) fibers as well as water-soluble poly(ethylene oxide) (PEO) fibers that serve as space holders during scaffold formation [4]. Removal of these PEO fibers prior to seeding resulted in improved cell infiltration after 3 weeks, but the long term maturation of such constructs has yet to be characterized. To assess the effect of sacrificial PEO fiber content on construct growth, a triple-jet electrospinning device was employed to generate PCL/PEO scaffolds with PEO fiber fractions ranging from 0 to 60%. After seeding with mesenchymal stem cells (MSCs), constructs were clamped in custom grips to maintain strip morphology. The mechanical and biochemical maturation of constructs was assessed over 9 weeks of free swelling culture in a chemically defined medium (CDM), along with cell infiltration and matrix distribution. We hypothesized that enhanced pore size in dual-fiber constructs would lead to not only a better distribution of cells, but also larger increases in stiffness resulting from enhanced matrix production and distribution.