Nanofiber size-dependent sensitivity of fibroblast directionality to the methodology for scaffold alignment
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.
Section snippets
Nanofiber electrospinning
All the chemicals were obtained from Sigma–Aldrich Co. (St Louis, MO) unless noted otherwise. Nanofibers of varying diameters were electrospun using PLGA with a lactic acid to glycolic acid ratio of 85:15 and a molecular weight of 50–70 kDa. The polymer was dissolved in a mixture of tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) at 20% w/v concentration. To reduce the diameter of the electrospun nanofibers to sizes from ∼800 to ∼100 nm, the dielectric constant of the solvent was gradually
Synthesis of size scaled mandrel aligned electrospun nanofibers
The dielectric strength of the solvent during electrospinning can be increased to produce smaller sizes of electrospun fibers. A higher dielectric strength solvent allows a higher degree of polarization of the jet during electrospinning, thereby enhancing stretching in the instability region and reducing the net electrospun nanofiber diameter at the collector. As shown in Fig. 1, upon increasing the dielectric strength of the solvent by increasing the proportion of DMF, the nanofiber size can
Discussion
The results in Fig. 7 suggest that on aligned scaffolds of large fibers (∼740 nm), cell directionality was not dependent on the methodology applied for nanofiber alignment. However, on aligned scaffolds of successively smaller fibers the cell directionality exhibits successively greater deviations from that of the underlying fiber alignment, especially on mandrel aligned fibers, where substantial deviations were observed on scaffolds with fibers sizes ⩽300 nm. With electrically aligned fibers, on
Conclusions
Fibroblast directionality on equivalently aligned nanofiber scaffolds was found to exhibit a strong dependence on the methodology applied for scaffold alignment, especially for scaffolds composed of successively smaller fiber sizes approaching 100 nm. On scaffolds of successively smaller fibers, the greater deviations in fibroblast directionality from that of the underlying fiber alignment is attributed to the higher likelihood of interaction of cell lamellipodia with multiple, rather than
Acknowledgements
This work was supported by NSF Grants 0701505 and 0902969 (to N.S.S.) and National Science Council (ROC) Grants 96-2112-M-001-024-MY3 and 99-2112-M-001-027-MY3 (to C.F.C.).
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