Influence of Touch-Spun Nanofiber Diameter on Contact Guidance during Peripheral Nerve Repair.

Peripheral nerve regeneration across large gaps remains clinically challenging and scaffold design plays a key role in nerve tissue engineering. One strategy to encourage regeneration has utilized nanofibers or conduits to exploit contact guidance within the neural regenerative milieu. Herein, we report the effect of nanofiber topography on two key aspects of regeneration: Schwann cell migration and neurite extension. Substrates possessing distinct diameter distributions (300 ± 40 to 900 ± 70 nm) of highly aligned poly(ε-caprolactone) nanofibers were fabricated by touch-spinning. Cell migratory behavior and contact guidance were then evaluated both at the tissue level using dorsal root ganglion tissue explants and the cellular level using dissociated Schwann cells. Explant studies showed that Schwann cells emigrated significantly farther on fibers than control. However, both Schwann cells and neurites emigrated from the tissue explants directionally along the fibers regardless of their diameter, and the data were characterized by high variation. At the cellular level, dissociated Schwann cells demonstrated biased migration in the direction of fiber alignment and exhibited a significantly higher biased velocity (0.2790 ± 0.0959 µm·min-1) on 900 ± 70 nm fibers compared to other nanofiber groups and similar to the velocity found during explant emigration on 900 nm fibers. Therefore, aligned, nanofibrous scaffolds of larger diameters (900 ± 70 nm) may be promising materials to enhance various aspects of nerve regeneration via contact guidance alone. While cells track along with the fibers, this contact guidance is bidirectional along the fiber, moving in the plane of alignment. Therefore, the next critical step to direct regeneration is to uncover haptotactic cues that enhance directed migration.

Postfabrication Tethering of Molecular Gradients on Aligned Nanofibers of Functional Poly(ε-caprolactone)s.

Substrates with combinations of topographical and biochemical cues are highly useful for a number of fundamental biological investigations. Tethered molecular concentration gradients in particular are highly desired for a number of biomedical applications including cell migration. Herein, we report a versatile method for the fabrication of aligned nanofiber substrates with a tunable concentration gradient along the fiber direction. 4-Dibenzocyclooctynol (DIBO) was used as an initiator for the ring-opening copolymerization of ε-caprolactone (εCL) and allyl-functionalized ε-caprolactone (AεPCL), which yielded a well-defined polymer with orthogonal functional handles. These materials were fabricated into aligned nanofiber substrates via touch-spinning. Fibers were modified post-spinning with a concentration gradient of fluorescently labeled dye via a light activated thiol-ene reaction through a photomask. As a demonstration, the cell adhesive peptide RGD was chemically tethered to the fiber surface at a second functionalization site via strain-promoted azide-alkyne cycloaddition (SPAAC). This novel approach affords fabrication of dual functional nanofiber substrates.

RGD-Functionalized Nanofibers Increase Early GFAP Expression during Neural Differentiation of Mouse Embryonic Stem Cells.

Stem cell differentiation toward a specific lineage is controlled by its microenvironment. Polymer scaffolds have long been investigated to provide microenvironment cues; however, synthetic polymers lack the specific signaling motifs necessary to direct cellular responses on their own. In this study, we fabricated random and aligned poly(ε-caprolactone) nanofiber substrates, surface-functionalized with RGD viastrain-promoted azide-alkyne cycloaddition, that were used to investigate the role of a covalently tethered bioactive peptide (RGD) and nanofiber orientation on neural differentiation of mouse embryonic stem cells. Gene and protein expression showed neural differentiation progression over 14 days, with similar expression on RGD random and aligned nanofibers for neurons and glia over time. The high levels of glial fibrillary acidic protein expression at early time points were indicative of neural progenitors, and occurred earlier than on controls or in previous reports. These results highlight the influence of RGD binding versus topography in differentiation.

A Comparative Study on the Effects of High-Intensity Focused Electromagnetic Technology and Electrostimulation for the Treatment of Pelvic Floor Muscles and Urinary Incontinence in Parous Women: Analysis of Posttreatment Data.

OBJECTIVES: Pelvic floor muscles (PFMs) weakening and urinary incontinence (UI) represent health issues that have a negative impact on daily life. This study compares the immediate efficiency of high-intensity focused electromagnetic (HIFEM) therapy and electrostimulation for the treatment of weakened PFMs, accompanied by the UI. METHODS: Ninety-five parous women were considered for the study. Symptomatic patients received either HIFEM or electrostimulation treatment. Treated patients completed 10 therapies scheduled 2 to 3 times per week (HIFEM) or every other day (electrostimulation). Patients underwent examination by 3-dimensional transperienal ultrasound at the baseline and posttreatments. Levator-urethra gap, anteroposterior diameter, laterolateral diameter of levator hiatus, and hiatal area were measured. In addition, Pelvic Floor Disability Index 20 questionnaire and subjective evaluation of patient's intimate health were assessed. RESULTS: Enrolled patients were divided into group I (n = 50, HIFEM), group II (n = 25, electrostimulation), and group III (n = 20, control) according the indication and treatment modality. Three-dimensional ultrasounds showed positive changes in dynamics of the pelvic floor posttreatment (decreased anteroposterior diameter, laterolateral diameter, and hiatal area). However, the significant (P < 0.05) changes of pelvic floor integrity were observed only in group I. In addition, group I achieved greater level of improvement in Pelvic Floor Disability Index 20 questionnaire compared with group II (52% and 18% respectively; P < 0.001). Substantially fewer patients in group I reported urine leakage after treatments. CONCLUSIONS: Posttreatment results suggest that HIFEM technology is suitable for treatment of PFMs weakening and showed to be more effective when compared with electrostimulation in short-term. Therefore, we recommend HIFEM as treatment option for weakened PFMs and UI.

RGD-Modified Nanofibers Enhance Outcomes in Rats after Sciatic Nerve Injury.

Nerve injuries requiring surgery are a significant problem without good clinical alternatives to the autograft. Tissue engineering strategies are critically needed to provide an alternative. In this study, we utilized aligned nanofibers that were click-modified with the bioactive peptide RGD for rat sciatic nerve repair. Empty conduits or conduits filled with either non-functionalized aligned nanofibers or RGD-functionalized aligned nanofibers were used to repair a 13 mm gap in the rat sciatic nerve of animals for six weeks. The aligned nanofibers encouraged cell infiltration and nerve repair as shown by histological analysis. RGD-functionalized nanofibers reduced muscle atrophy. During the six weeks of recovery, the animals were subjected to motor and sensory tests. Sensory recovery was improved in the RGD-functionalized nanofiber group by week 4, while other groups needed six weeks to show improvement after injury. Thus, the use of functionalized nanofibers provides cues that aid in in vivo nerve repair and should be considered as a future repair strategy.

Accelerated neural differentiation of mouse embryonic stem cells on aligned GYIGSR-functionalized nanofibers.

Substrates for embryonic stem cell culture are typified by poorly defined xenogenic, whole proteins or cellular components that are difficult and expensive to generate, characterize, and recapitulate. Herein, the generation of well-defined scaffolds of Gly-Tyr-Ile-Gly-Ser-Arg (GYIGSR) peptide-functionalized poly(ε-caprolactone) (PCL) aligned nanofibers are used to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells (mESCs). Gene expression trends and immunocytochemistry analysis were similar to laminin-coated glass, and indicated an earlier differentiation progression than D3 mESCs on laminin. Further, GYIGSR-functionalized nanofiber substrates yielded an increased gene expression of Sox1, a neural progenitor cell marker, and Tubb3, Cdh2, Syp, neuronal cell markers, at early time points. In addition, guidance of neurites was found to parallel the fiber direction. We demonstrate the fabrication of a well-defined, xeno-free functional nanofiber scaffold and demonstrates its use as a surrogate for xenogenic and complex matrixes currently used for the neural differentiation of stem cells ex vivo. STATEMENT OF SIGNIFICANCE: In this paper, we report the use of GYIGSR-functionalized poly(ε-caprolactone) aligned nanofibers as a tool to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells. The results indicate that functional nanofiber substrates promote faster differentiation than laminin coated substrates. The data suggest that aligned nanofibers and post-electrospinning surface modification with bioactive species can be combined to produce translationally relevant xeno-free substrates for stem cell therapy. Future development efforts are focused on additional bioactive species that are able to function as surrogates for other xenogenic factors found in differentiation media.