Poly-l-lactic acid-co-poly(pentadecalactone) Electrospun Fibers Result

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Poly-L-lactic acid-co-poly(pentadecalactone) electrospun fibers result in greater neurite outgrowth of chick dorsal root ganglia in vitro compared to poly-L-lactic acid fibers Alexis M Ziemba, Keith P Lane, Ignacio M San Segundo, Anthony R D'Amato, Andrew K Mason, Ryan J Sexton, Hubert Casajus, Richard A Gross, David T. Corr, and Ryan J. Gilbert ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00013 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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Poly-L-lactic acid-co-poly(pentadecalactone) electrospun fibers result in greater neurite outgrowth of chick dorsal root ganglia in vitro compared to poly-L-lactic acid fibers AUTHOR NAMES: Alexis M. Ziemba1,3, Keith P. Lane1,3, Ignacio M. San Segundo2†, Anthony R. D’Amato1,3, Andrew K. Mason1, Ryan J. Sexton1, Hubert Casajus2ǂ, Richard A. Gross2,3, David T. Corr1, Ryan J. Gilbert1,3* AUTHOR ADDRESS: 1Department of Biomedical Engineering, 110 8th Street, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; 2Department of Chemistry and Chemical Biology, 110 8th Street, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA; 3Center for Biotechnology and Interdisciplinary Sciences, 1623 15th Street, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA Present Addresses † Center for Nanomedicine and Theranostics, Department of Chemistry, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark. ǂ Ecole Nationale Supérieure de Chimie de Rennes, 11 Allée de Beaulieu, 35708 Rennes, France. Corresponding Author *E-mail: [email protected].

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ORCID Ryan J. Gilbert: 0000-0002-3501-6753, Richard A. Gross: 0000-0002-5050-3162, Alexis M. Ziemba: 0000-0001-8947-9617

KEYWORDS: electrospun fibers, mechanical property, surface topography, dorsal root ganglia, neurite outgrowth, neural engineering

ABSTRACT: Electrospun poly-L-lactic acid (PLLA) fiber scaffolds are used to direct axonal extension in neural engineering models. We aimed to improve the efficacy of these fibers in promoting neurite outgrowth by altering surface topography and reducing fiber elastic modulus through the incorporation of a compatibilized blend, poly-L-lactic acid-poly(pentadecalactone) (PLLA-PPDL) into the solution prior to electrospinning. PLLA+PLLA-PPDL fibers had a larger diameter, increased surface nanotopography, and lower glass transition temperature than PLLA fibers but had similar mechanical properties. Increases in neurite outgrowth on PLLA+PLLAPPDL fibers were observed, potentially due to the significantly increased diameter and surface coverage with nanotopography. Ultimately, these results suggest that greater electrospun fiber diameter and surface topography may contribute to increases in neurite outgrowth.

TEXT Electrospun fibers are commonly used for tissue engineering purposes as the nanofiber topography mimics that of the extracellular matrix1–3. Electrospun fibers are particularly beneficial for neural engineering applications; for example, spinal cords are composed of bundles of myelinated axons that run longitudinally and thus require anisotropic guidance to bridge lesion gaps4,5. Aligned electrospun fibers provide directional guidance, enhancing


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extension of dorsal root ganglia (DRG) neurites in vitro compared to randomly-oriented fibers6–8 and producing a nearly 2-fold greater axonal regeneration compared to randomly-oriented fibers and films within a rat T9-10 transection injury6. Similarly, electrospun fiber scaffolds enable regeneration in peripheral nerve injury models as alternatives to autographs and allographs9. Electrospun fiber conduits increase myelinated axons in regenerating rat sciatic nerves10 in addition to reconnecting sciatic nerves and re-innervating muscles in a rat sciatic transection injury model11. These aligned fiber scaffolds hold great promise for nerve regeneration; however, their surface and mechanical properties should be optimized to improve regenerative efficacy. Electrospun fiber scaffolds with porous surfaces are engineered by electrospinning common polymers, such as poly(ɛ-caprolactone) (PCL) and poly-L-lactic acid (PLLA), with other polymers or non-solvents that are later removed via dissolution or evaporation12,13. To date, primary neurons have not been studied on electrospun fibers with varying surface topographies. However, a study by Fozdar et al. demonstrated that rat hippocampal cells prefer quartz surfaces with patterned topography over smooth surfaces, particularly 300 nm holes14. Similarly, PC12 cells (nerve growth factor-responsive cells) migrated preferentially onto nanoporous regions of SU-8 resin substrates15. These studies suggest that fibers with surface nanotopography may be more supportive of neurite outgrowth. In addition to surface topography, mechanical properties should also be considered when fabricating electrospun fibers for neural interfacing. Hydrogels with a lower modulus (low kPa range) are more supportive of neuronal differentiation, neurite outgrowth, and neuronal adhesion16–18; however, fiber moduli are typically orders of magnitude higher. Tensiometry tests have shown that 250-750 nm diameter electrospun PCL fibers have a mean modulus of 3.7 GPa19, while neat extruded PLLA has a modulus of greater than one GPa20. Ultimately, current


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fiber moduli are greater than native tissue moduli21, so reducing fiber moduli may increase the regenerative efficacy of these fiber scaffolds. As PLLA fibers have demonstrated efficacy in promoting SCI regeneration6,22 and have been well characterized in our lab, we aimed to improve the surface and mechanical properties of PLLA fibers to increase regenerative efficacy. Spinella et al. previously demonstrated that the reactive blending of poly(ω-hydroxytetradecanoic acid) with PLLA results in a PLLA blend with a reduced modulus23. Since the structure of poly(ω-pentadecalactone) (PPDL) is almost identical to poly(ω-hydroxytetradecanoic acid) and PPDL is more ductile than PLLA24, reactive blends of PLLA and PPDL were prepared using the method published by Spinella et al. to give PLLAPPDL. This blending strategy avoids risks associated with common plasticizers that leach out of medical materials, in some cases act as xenoestrogens, and cause endocrine disruption25,26. In this study, the compatibilized blend, PLLA-PPDL, was produced by reactive extrusion (please refer to Supporting Information for all detailed methods). PLLA-only and PLLA+PLLA-PPDL fibers were electrospun using previously described methods6,13,27,28. Fibers were characterized using scanning electron microscopy (SEM) for physical characteristics, contact angle for surface wettability, differential scanning calorimetry (DSC) for thermal characteristics, and single fiber tensile testing for mechanical properties. To determine the effect of PLLA-PPDL on neurite outgrowth, embryonic day nine (E9) chick dorsal root ganglia (DRG) were cultured on both fiber types for five days in vitro, and neurite outgrowth was measured. We hypothesized that the addition of PLLA-PPDL would reduce fiber modulus, enhance surface topography, and ultimately increase neurite outgrowth in vitro.


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Fiber diameter, alignment, density, and morphology for PLLA (Figure 1A) and PLLA+PLLAPPDL (Figure 1B) were first studied using SEM and ImageJ analysis. The mean diameter of PLLA fibers was 1.75 ± 0.74 µm while PLLA+PLLA-PPDL fibers (in regions without protrusions) were significantly larger in diameter, 2.20 ± 0.80 µm (T[512] = -6.58, p = 0.000; Figure 1C). The increase in PLLA+PLLA-PPDL fiber diameter is likely a result of the increase in polymer concentration (8% to 10%) necessary for fiber formation, as PLLA-PPDL has a lower molecular weight than PLLA. Electrospun fiber diameter affects the amount of axonal extension of neurons and the final lineage to which neural stem cells differentiate27,29. Numerous studies have shown that neurites extend the furthest along fibers with diameters in the low micron range (~800 nm to five µm)27,30, suggesting diameters in this range enhance neurite sensing of and guidance by fiber topography. For fiber alignment, 94.1% of PLLA fibers were aligned within 5° of the central axis while 91.1% of PLLA+PLLA-PPDL fibers were aligned within 5° of the central axis (T[520] = -0.30, p = 0.76; Figure 1D). As previously described, anisotropic guidance increases neurite outgrowth6–8; as the majority of fibers were within 5° of the central axis, alignment did not affect neurite outgrowth in this study. For fiber density, PLLA fibers covered 62.6 ± 10.9 % of the surface of 7021 µm2 regions while PLLA+PLLA-PPDL fibers covered 65.7 ± 19.2 % of the surface (T[27] = -0.57, p = 0.57; Figure 1E). A study by Xie et al. previously demonstrated that high collection density (15 min collection) fibers result in increased perpendicular neurite outgrowth compared to lower collection density (4 min collection) electrospun fibers31; however, neither fiber type was dense enough to provoke perpendicular outgrowth in this study. As alignment and density are comparable for both fiber types, these characteristics are not likely the cause of changes in neurite outgrowth observed here.


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Figure 1: PLLA+PLLA-PPDL fibers have significantly larger fiber diameter than PLLA fibers. Sample SEM images of A) PLLA fibers and B) PLLA+PLLA-PPDL fibers, scale bar 20 µm. C) Summary graph of fiber diameter of PLLA and PLLA+PLLA-PPDL fibers. Data are represented as mean ± standard deviation. Asterisk indicates a statistically significant difference in fiber diameter by two-sample t-test, **p

Poly-l-lactic acid-co-poly(pentadecalactone) Electrospun Fibers Result

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