Influence of oriented nanofibrous PCL scaffolds on quantitative gene expression during neural differentiation of mouse embryonic stem cells.

Neural differentiation of mouse embryonic stem cells in combination with three-dimensional electrospun nanofibers as an artificial extracellular matrix can be utilized to reconstruct a spinal cord defect. In this study, random and parallel-aligned nanofibrous poly ɛ-caprolactone was fabricated using electrospinning. Its hydrophobicity was modified by O2 plasma treatment to facilitate enhanced cell attachment. Embryoid bodies (EBs), which contain all three embryonic germ layers, were cultured on poly ɛ-caprolactone scaffolds to study the effect of fiber orientation on cell morphology and differentiation. Cell morphology and neuron-specific gene and protein expressions were, respectively, evaluated by scanning electron microscopy, real-time polymerase chain reaction, and immunocytochemistry. Although two types of nanofibrous scaffolds showed neural marker expression at the protein level, cells on randomly oriented scaffolds showed short-range topographical guidance and stretched across multiple directions, whereas cells on the parallel scaffolds exhibited long extension with enhanced neuron outgrowth along the fiber, producing oriented extracellular matrix, leading to direct cell migration and nerve regeneration. Quantitative real-time polymerase chain reaction showed that both aligned and random electrospun nanofibers downregulated the precursor neural marker Nestin compared with that in the control group, a gelatin-coated tissue culture plate (T). Analysis also showed higher expression of dorso-ventral neural markers (Isl1/2 and Lim1/2) than motor neuron progenitor markers (Pax6, Nkx6.1, and olig2) in aligned nanofibers than in the T group. Moreover, aligned nanofibers showed higher expression of mature neural specific markers such as ß-tub and Map2 than those in the randomly oriented scaffolds. Therefore, we conclude that nanofibers with different orientations can support the neural lineage, but aligned nanofibrous scaffolds are superior candidates to promote the advancement of neural precursors to achieve maturity during the differentiation process.

Controlled surface morphology and hydrophilicity of polycaprolactone toward selective differentiation of mesenchymal stem cells to neural like cells.

Differentiation of mesenchymal stem cells (MSCs) into neuron cells has great potential in therapy of damaged nerve tissue. It has been shown that three-dimensional biomaterials have great ability to up regulate the expression of neuronal proteins. In this study, O2 plasma technology was used to enhance hydrophilicity of poly (ε-caprolactone) (PCL) toward selective differentiation of MSCs into neural cells. Random and aligned PCL nanofibers scaffolds were fabricated by electrospinning method and their physicochemical and mechanical properties were carried out by scanning electron microscope (SEM), contact angle, and tensile measurements. Contact angle studies of PCL and plasma treated PCL (p-PCL) nanofibers revealed significant change on the surface properties PCL nanofibers from the view point of hydrophilicity. Physiochemical studies revealed that p-PCL nanofibers were extremely hydrophilic compared with untreated PCL nanofibers which were highly hydrophobic and nonabsorbent to water. Differentiation of MSCs were carried out by inducing growth factors including basic fibroblast growth factor, nerve growth factor, and brain derived growth factor, NT3, 3-isobutyl-1-methylxanthine (IBMX) in Dulbecco's modified Eagle's medium/F12 media. Differentiated MSCs on nanofibrous scaffold were examined by immunofluorescence assay and was found to express the neuronal proteins; ß-tubulin III and Map2, on day 15 after cell culture. The real-time polymerase chain reaction (RT-PCR) analysis showed that p-PCL nanofibrous scaffold could upregulate expression of Map-2 and downregulate expression of Nestin genes in nerve cells differentiated from MSCs. This study indicates that mesenchymal stem cell cultured on nanofibrous scaffold have potential differentiation to neuronal cells on and could apply in nerve tissue repair.

The effect of aligned and random electrospun fibrous scaffolds on rat mesenchymal stem cell proliferation.

OBJECTIVE: The development of combining mesenchymal stem cells (MSCs) with surface modified three-dimensional (3D) biomaterial scaffold provides a desirable alternative for replacement of damaged and diseased tissue. Nanofibrous scaffolds serve as suitable environment for cell attachment and proliferation due to their similarity to the physical dimension of the natural extracellular matrix. In this study the properties of plasma treated poly-C-caprolactone nanofiber scaffolds (p-PCL) and unaltered PCL scaffolds were compared, and then p-PCL scaffolds were evaluated for MSC culture. MATERIALS AND METHODS: Aligned and random PCL nanofibrus scaffolds were fabricated by electrospining and their surface modified with O2 plasma treatment to enhance MSC proliferation, adhesion and interaction. Chemical and mechanical characterizations were carried out using scanning electron microscopy (SEM), water contact angle and tensile testing. Cell adhesion and morphology were evaluated using SEM 1 day after culture. Statistical analysis was carried out using one way analysis of variance (ANOVA). RESULTS: The proliferation of MSCs were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide(MTT) assay on day 1, 3, and 5 after cell culture. Results showed that the numbers of cells that had grown on PCL nanofibrous scaffolds were significantly higher than those of control surfaces without nanofibers. Furthermore, the proliferation of MSCs on random nanofiber was significantly higher compared to that on aligned nanofiber. CONCLUSION: This study showed that while both aligned and random plasma treated PCL nanofibrous scaffold are more suitable substrates for MSC growth than tissue culture plates, random nanofiber best supported the proliferation of MSCs.