Electrospinning is a promising approach to create nanofiber structures that are capable of supporting adhesion and guiding extension of neurons for nerve regeneration. Concurrently, electrical stimulation of neurons in the absence of topographical features also has been shown to guide axonal extension. Therefore, the goal of this study was to form electrically conductive nanofiber structures and to examine the combined effect of nanofiber structures and electrical stimulation. Conductive meshes were produced by growing polypyrrole (PPy) on random and aligned electrospun poly(lacticco-glycolic acid) (PLGA) nanofibers, as confirmed by scanning electron micrographs and X-ray photon spectroscopy. PPy-PLGA electrospun meshes supported the growth and differentiation of rat pheochromocytoma 12 (PC12) cells and hippocampal neurons comparable to non-coated PLGA control meshes, suggesting that PPy-PLGA may be suitable as conductive nanofibers for neuronal tissue scaffolds. Electrical stimulation studies showed that PC12 cells, stimulated with a potential of 10 mV/cm on PPy-PLGA scaffolds, exhibited 40-50% longer neurites and 40-90% more neurite formation compared to unstimulated cells on the same scaffolds. In addition, stimulation of the cells on aligned PPy-PLGA fibers resulted in longer neurites and more neurite-bearing cells than stimulation on random PPy-PLGA fibers, suggesting a combined effect of electrical stimulation and topographical guidance and the potential use of these scaffolds for neural tissue applications.
Effective strategies to guide cell alignment and the deposition of an oriented extracellular matrix are critical for the development of anisotropic engineered tissues suitable for the repair of ligament defects. Electrospinning is a promising means to create meshes that can align adherent cells, but the effect of fiber mesh architecture on differentiation has not been examined closely. Therefore, the goal of this study was to determine the effect of fiber diameter and the degree of fiber alignment on mesenchymal progenitor cell morphology, proliferation, and ligament gene expression. Specifically, a poly(ester urethane)urea elastomer was electrospun onto rigid supports under conditions designed to independently vary the mean fiber diameter (from 0.28 to 2.3 microm) and the degree of fiber alignment. Bone marrow stromal cells--seeded onto supported meshes--adhered to and proliferated on all surfaces. Cells assumed a more spindle-shaped morphology with increasing fiber diameter and degree of fiber alignment, and oriented parallel to fibers on aligned meshes. Expression of the ligament markers collagen 1alpha1, decorin, and tenomodulin appeared to be sensitive to fiber diameter and greatest on the smallest fibers. Concurrently, expression of the transcription factor scleraxis appeared to decrease with increasing fiber alignment. These results suggest that the formation of a ligament-like tissue on electrospun scaffolds is enhanced when the scaffolds consist of aligned submicron fibers.
The mechanical properties of biomaterial scaffolds are crucial for their efficacy in tissue engineering and regenerative medicine. At the microscopic scale, the scaffold must be sufficiently rigid to support cell adhesion, spreading, and normal extracellular matrix deposition. Concurrently, at the macroscopic scale the scaffold must have mechanical properties that closely match those of the target tissue. The achievement of both goals may be possible by careful control of the scaffold architecture. Recently, electrospinning has emerged as an attractive means to form fused fibre scaffolds for tissue engineering. The diameter and relative orientation of fibres affect cell behaviour, but their impact on the tensile properties of the scaffolds has not been rigorously characterized. To examine the structure-property relationship, electrospun meshes were made from a polyurethane elastomer with different fibre diameters and orientations and mechanically tested to determine the dependence of the elastic modulus on the mesh architecture. Concurrently, a multiscale modelling strategy developed for type I collagen networks was employed to predict the mechanical behaviour of the polyurethane meshes. Experimentally, the measured elastic modulus of the meshes varied from 0.56 to 3.0 MPa depending on fibre diameter and the degree of fibre alignment. Model predictions for tensile loading parallel to fibre orientation agreed well with experimental measurements for a wide range of conditions when a fitted fibre modulus of 18 MPa was used. Although the model predictions were less accurate in transverse loading of anisotropic samples, these results indicate that computational modelling can assist in design of electrospun artificial tissue scaffolds.
Engineered scaffolds simultaneously exhibiting multiple cues are highly desirable for neural tissue regeneration. To this end, we developed a neural tissue engineering scaffold that displays submicrometer-scale features, electrical conductivity, and neurotrophic activity. Specifically, electrospun poly(lactic acid-co-glycolic acid) (PLGA) nanofibers were layered with a nanometer thick coating of electrically conducting polypyrrole (PPy) presenting carboxylic groups. Then, nerve growth factor (NGF) was chemically immobilized onto the surface of the fibers. These NGF-immobilized PPy-coated PLGA (NGF-PPyPLGA) fibers supported PC12 neurite formation (28.0±3.0% of the cells) and neurite outgrowth (14.2 µm median length), which were comparable to that observed with NGF (50 ng/mL) in culture medium (29.0±1.3%, 14.4 µm). Electrical stimulation of PC12 cells on NGF-immobilized PPyPLGA fiber scaffolds was found to further improve neurite development and neurite length by 18% and 17%, respectively, compared to unstimulated cells on the NGF-immobilized fibers. Hence, submicrometer-scale fibrous scaffolds that incorporate neurotrophic and electroconducting activities may serve as promising neural tissue engineering scaffolds such as nerve guidance conduits.
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