Yee-Shuan Lee

Neurite extension of primary neurons on electrospun piezoelectric scaffolds

Neural tissue engineering may be a promising option for neural repair treatment, for which a well-designed scaffold is essential. Smart materials that can stimulate neurite extension and outgrowth have been investigated as potential scaffolding materials. A piezoelectric polymer polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) was used to fabricate electrospun aligned and random scaffolds having nano- or micron-sized fiber dimensions. The advantage of using a piezoelectric polymer is its intrinsic electrical properties. The piezoelectric characteristics of PVDF-TrFE scaffolds were shown to be enhanced by annealing. Dorsal root ganglion (DRG) neurons attached to all fibrous scaffolds. Neurites extended radially on random scaffolds, whereas aligned scaffolds directed neurite outgrowth for all fiber dimensions. Neurite extension was greatest on aligned, annealed PVDF-TrFE having micron-sized fiber dimensions in comparison with annealed and as-spun random PVDF-TrFE scaffolds. DRG on micron-sized aligned, as-spun and annealed PVDF-TrFE also had the lowest aspect ratio amongst all scaffolds, including non-piezoelectric PVDF and collagen-coated substrates. Findings from this study demonstrate the potential use of a piezoelectric fibrous scaffold for neural repair applications.

Characterization and in vitro cytocompatibility of piezoelectric electrospun scaffolds.

Previous studies have shown that electrical charges influence cell behavior (e.g. enhancement of nerve regeneration, cell adhesion, cell morphology). Thus, piezoelectric scaffolds might be useful for various tissue engineering applications. Fibrous scaffolds were successfully fabricated from permanent piezoelectric poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE) by the electrospinning technique. Scanning electron microscopy and capillary flow analyses verified that the fiber mats had an average fiber diameter of 970 +/- 480 nm and a mean pore diameter of 1.7 microm, respectively. Thermally stimulated depolarization current spectroscopy measurements confirmed the piezoelectric property of the PVDF-TrFE fibrous scaffolds by the generation of a spontaneous current with the increase in temperature in the absence of an electric field, which was not detected in the unprocessed PVDF-TrFE powder. Differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction and Fourier transform infrared spectroscopy results showed that the electrospinning process increased the crystallinity and presence of the polar, beta-phase crystal compared with the unprocessed powder. Confocal fluorescence microscopy and a cell proliferation assay demonstrated spreading and increased cell numbers (human skin fibroblasts) over time on PVDF-TrFE scaffolds, which was comparable with tissue culture polystyrene. The relative quantity of gene expression for focal adhesion proteins (measured by real-time RT-PCR) increased in the following order: paxillin < vinculin < focal adhesion kinase < talin. However, no differences could be seen among the TCPS surface and the fibrous scaffolds. Future studies will focus on possible applications of these cytocompatible PVDF-TrFE scaffolds in the field of regenerative medicine.

Mesenchymal stem cells accelerate bone allograft incorporation in the presence of diabetes mellitus

Allograft (Allo) incorporation in the presence of a systemic disease like diabetes mellitus (DM) is becoming a major issue in the orthopedic community. Mesenchymal stem cells (MSC) are multipotent stem cells that may be derived from adult, whole bone marrow and have been shown to induce bone formation in segmental defects when combined with the appropriate carrier/scaffold. The objectives of this study were to analyze the effect of DM upon Allo incorporation in a segmental rat femoral defect and to also investigate MSC augmentation of Allo incorporation. Segmental (5 mm) femoral defects were created in non‐DM and DM rats and treated with Allo containing demineralized bone matrix (DBM) or DBM with MSC augmentation. Histological scoring at 4 weeks demonstrated less mature bone in the DM/DBM group compared to its non‐DM counterpart (p < 0.001). However, there was significantly more mature bone in the DM/MSC group when compared to the DM/DBM group at both 4 and 8 weeks (p < 0.001 and p = 0.004). Furthermore, significantly more bone formation was observed in the DM/MSC group compared to the DM/DBM group at the 4‐week time point (p < 0.001). The results of this study suggest that MSC are a potential adjunct for bone regeneration when implanted in an orthotopic site in the presence of DM.

Aligned fibrous PVDF-TrFE scaffolds with Schwann cells support neurite extension and myelination in vitro

OBJECTIVE Polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE), which is a piezoelectric, biocompatible polymer, holds promise as a scaffold in combination with Schwann cells (SCs) for spinal cord repair. Piezoelectric materials can generate electrical activity in response to mechanical deformation, which could potentially stimulate spinal cord axon regeneration. Our goal in this study was to investigate PVDF-TrFE scaffolds consisting of aligned fibers in supporting SC growth and SC-supported neurite extension and myelination in vitro. APPROACH Aligned fibers of PVDF-TrFE were fabricated using the electrospinning technique. SCs and dorsal root ganglion (DRG) explants were co-cultured to evaluate SC-supported neurite extension and myelination on PVDF-TrFE scaffolds. MAIN RESULTS PVDF-TrFE scaffolds supported SC growth and neurite extension, which was further enhanced by coating the scaffolds with Matrigel. SCs were oriented and neurites extended along the length of the aligned fibers. SCs in co-culture with DRGs on PVDF-TrFE scaffolds promoted longer neurite extension as compared to scaffolds without SCs. In addition to promoting neurite extension, SCs also formed myelin around DRG neurites on PVDF-TrFE scaffolds. SIGNIFICANCE This study demonstrated PVDF-TrFE scaffolds containing aligned fibers supported SC-neurite extension and myelination. The combination of SCs and PVDF-TrFE scaffolds may be a promising tissue engineering strategy for spinal cord repair.