Nanofiber size-dependent sensitivity of fibroblast directionality to the methodology for scaffold alignment

Abstract

The sensitivity of fibroblast guidance on directional cues provided by aligned nanofibers is studied for scaffolds of successively smaller fiber sizes (740 ± 280, 245 ± 85, 140 ± 40, and 80 ± 10 nm) fabricated using mandrel and electrical alignment methodologies for electrospun nanofibers (∼10° angular deviation (AD)), as well as nanoimprint methodologies for perfectly aligned fibers (0° AD). On aligned scaffolds of large fibers (∼740 nm) cell directionality closely follows the underlying fibers, irrespective of the alignment method. However, on mandrel aligned scaffolds of successively smaller fibers the cell directionality exhibits greater deviations from the underlying fiber alignment due to the higher likelihood of interaction of cell lamellipodia with multiple, rather than single, nanofibers. Using electrically aligned scaffolds, fibroblast directionality deviations can be maintained in the range of nanofiber alignment deviation for fiber sizes down to ∼100 nm. This improvement in cell guidance is attributed to molecular scale directional adhesion cues for cell receptors, which occur within electrically aligned scaffolds due to fiber polarization parallel to the geometric alignment axis of the nanofiber under the modified electric field during electrospinning. While fibroblast directionality is similar on electrically aligned vs. nanoimprinted scaffolds for fiber sizes >100 nm, cell directionality is influenced more strongly by the perfect alignment cues of the latter on ∼100 nm fiber scaffolds. The scaffold alignment methodology is hence highly significant, especially for tissue engineering applications requiring sub-100 nm aligned fibers.

Introduction

Aligned nanofiber scaffolds are commonly applied in tissue engineering to provide directional cues for cell migration and to enhance regeneration of oriented tissues, such as peripheral nerve and ligament tissues [1], [2], [3], [4], [5]. Scaffolds composed of nanofibers of successively smaller size promote various functionalities of the cells, such as the expression of ligament-specific biomarkers for mesenchymal progenitor cells [6], enabling better cell differentiation and proliferation of stem cells [7] and improving the adhesion and growth kinetics of fibroblasts [8], [9]. Although there is a general trend towards studying cell and tissue interactions on progressively smaller nanofibers, the impact of the degree of alignment of size scaled fibers has been more difficult to assess. Previous studies of fibroblast cell guidance on polymeric nanofiber scaffolds composed of poly(d,l-lactic-co-glycolic acid) (PLGA) [10] and poly(methyl methacrylate) (PMMA) [11] suggest that for equivalently aligned nanofibers of sizes <1 μm, nanofibers below a critical size (∼700–1000 nm) were unable to provide effective guidance cues to cells. This may be attributed to the relatively large size of focal adhesion complexes [12], [13] in comparison with the fiber size, resulting in weaker complexes due to the availability of fewer anchor points for cell adhesion on scaffolds of smaller nanofibers. However, recent work has demonstrated that even on nanopatterned surfaces containing fewer anchor points, the presence of finely spaced anchor points (<90 nm) can promote cell adhesion through effective recruitment of integrin proteins to enable the formation of more stable focal adhesion complexes [14], [15]. Herein we aim to understand the role of the method of fabrication of aligned nanofiber scaffolds on their guidance characteristics. Specifically, do the highly directional cues provided by perfectly aligned nanoimprinted fibers (0° angular deviation (AD)) and molecular scale fiber polarization cues of electrically aligned electrospun fibers (∼10° AD) enhance cell guidance, especially on smaller sized nanofibers approaching ∼100 nm, presumably through promoting conditions for the formation of more stable focal adhesion sites.

Nanofiber alignment is usually enabled by rotating mandrel-based mechanical approaches [16] or by electrical approaches for alignment based on field modification using insulator gaps [17] or dielectrics [18], [19] patterned on the collector. While nanofibers can be geometrically aligned (as defined by small angular deviations of the fibers) over large areas (a few square centimeters) by mandrel-based mechanical approaches, they confer only a limited degree of molecular level orientation of the polymer functional groups on nanofibers in comparison with fibers polarized under a patterned electrical field [20]. The influence of this molecular level dipole orientation on the cell guidance characteristics, especially for sub-500 nm fibers, which are less effective in guiding cell directionality than larger fibers [10], [11], has not been studied previously. A chief challenge is the alignment of smaller sized electrospun nanofibers to a high degree, since they experience larger distortions arising from their greater flux and their longer time within the instability region [21]. Through optimization of the insulator gap width to enhance the spatial extent of electrical alignment forces we have fabricated size scaled fibers down to sub-100 nm sizes, aligned at ∼10° AD from the average direction [22], with a highly directional fiber polarization that likely causes orientation of functional groups along the polymer structure [20]. Additionally, we have optimized mandrel methods to enable nanofiber alignment at ∼10° AD for fiber sizes down to ∼100 nm, while preventing the breakage of smaller fibers at high rotational speeds through the use of a surfactant to reduce surface tension. To enhance the influence of aligned electrospun nanofibers on cell guidance, we have reduced the interactions of cells with the underlying substrate by collecting aligned fibers on Mylar® (polyethylene terephthalate resin plastic sheets) rather than coverslip glass substrates for cell culture, since the cells interact with glass but not with Mylar®. Finally, based on prior work on micro- [23] and nanoimprinted gratings [24], [25], we have recently developed a technique to pattern bio-compatible PLGA, so that the fibroblast guidance characteristics on these perfectly aligned nanofibers can be compared with equivalently sized electrospun PLGA nanofibers aligned by mandrel and electrical approaches. In this manner, through comparisons of highly aligned electrospun fibers (∼10° AD) with perfectly aligned nanoimprinted fibers (0° AD) of equivalent size, we are able to independently gauge the influence of geometric alignment over large lengths (using mandrel methods) and directional fiber polarization at the molecular level (using electrical methods) on the respective fibroblast guidance characteristics.