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The Effect of Electrospun Gelatin Fibers Alignment on Schwann Cell and Axon Behavior and Organization in the Perspective of Artificial Nerve Design.

Gnavi S, Fornasari BE, Tonda-Turo C, Laurano R, Zanetti M, Ciardelli G, Geuna S - Int J Mol Sci (2015)

Bottom Line: Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers.B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers.The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043, Italy. sara.gnavi@unito.it.

ABSTRACT
Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

No MeSH data available.


Related in: MedlinePlus

Adhesion assay: Confocal images (63× magnification) after DAPI (blue), tetramethylrhodamine (TRITC)-conjugated phalloidin (red) and vinculin (green) staining of RT4-D6P2T (A) and primary SC (B) on poly-l-lysine coated coverslips (control condition), random fibers and aligned fibers 3 h after seeding. Scale bar: 40 μm; RT4-D6P2T (C) and primary SC (D) cell numbers were expressed as cells/mm2 ± standard error of the mean (SEM). Statistical analysis was carried out using one-way ANOVA. Asterisks refer to significant statistical difference with *p ≤ 0.05 and ***p ≤ 0.001.
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ijms-16-12925-f002: Adhesion assay: Confocal images (63× magnification) after DAPI (blue), tetramethylrhodamine (TRITC)-conjugated phalloidin (red) and vinculin (green) staining of RT4-D6P2T (A) and primary SC (B) on poly-l-lysine coated coverslips (control condition), random fibers and aligned fibers 3 h after seeding. Scale bar: 40 μm; RT4-D6P2T (C) and primary SC (D) cell numbers were expressed as cells/mm2 ± standard error of the mean (SEM). Statistical analysis was carried out using one-way ANOVA. Asterisks refer to significant statistical difference with *p ≤ 0.05 and ***p ≤ 0.001.

Mentions: RT4-D6P2T and primary SC cultures were seeded on control condition (polylysine coated coverslips), gelatin random fibers and aligned fibers. After 3 h, the adherent cells were counted and their morphology examined. Figure 2 shows the effect of fiber alignment on cell adhesion and morphology. The alignment of gelatin electrospun fibers affected the number of adherent cells for RT4-D6P2T (p < 0.05) (Figure 2C) and primary SC (p < 0.001) (Figure 2D) cultures. When seeded on aligned fibers, there was less adhesion than under control conditions or random fibers. Both RT4-D6P2T and primary SC had high actin cytoskeleton organization and many focal adhesion points under all conditions tested. Its staining showed that both RT4-D6P2T and primary SC cultured on aligned fibers had elongated actin fibers compared to the control condition and random fibers (Figure 2A). Finally, cells had an elongated morphology with their longitudinal axis parallel to the direction of the aligned gelatin nano-fibers (Figure 2A).


The Effect of Electrospun Gelatin Fibers Alignment on Schwann Cell and Axon Behavior and Organization in the Perspective of Artificial Nerve Design.

Gnavi S, Fornasari BE, Tonda-Turo C, Laurano R, Zanetti M, Ciardelli G, Geuna S - Int J Mol Sci (2015)

Adhesion assay: Confocal images (63× magnification) after DAPI (blue), tetramethylrhodamine (TRITC)-conjugated phalloidin (red) and vinculin (green) staining of RT4-D6P2T (A) and primary SC (B) on poly-l-lysine coated coverslips (control condition), random fibers and aligned fibers 3 h after seeding. Scale bar: 40 μm; RT4-D6P2T (C) and primary SC (D) cell numbers were expressed as cells/mm2 ± standard error of the mean (SEM). Statistical analysis was carried out using one-way ANOVA. Asterisks refer to significant statistical difference with *p ≤ 0.05 and ***p ≤ 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490479&req=5

ijms-16-12925-f002: Adhesion assay: Confocal images (63× magnification) after DAPI (blue), tetramethylrhodamine (TRITC)-conjugated phalloidin (red) and vinculin (green) staining of RT4-D6P2T (A) and primary SC (B) on poly-l-lysine coated coverslips (control condition), random fibers and aligned fibers 3 h after seeding. Scale bar: 40 μm; RT4-D6P2T (C) and primary SC (D) cell numbers were expressed as cells/mm2 ± standard error of the mean (SEM). Statistical analysis was carried out using one-way ANOVA. Asterisks refer to significant statistical difference with *p ≤ 0.05 and ***p ≤ 0.001.
Mentions: RT4-D6P2T and primary SC cultures were seeded on control condition (polylysine coated coverslips), gelatin random fibers and aligned fibers. After 3 h, the adherent cells were counted and their morphology examined. Figure 2 shows the effect of fiber alignment on cell adhesion and morphology. The alignment of gelatin electrospun fibers affected the number of adherent cells for RT4-D6P2T (p < 0.05) (Figure 2C) and primary SC (p < 0.001) (Figure 2D) cultures. When seeded on aligned fibers, there was less adhesion than under control conditions or random fibers. Both RT4-D6P2T and primary SC had high actin cytoskeleton organization and many focal adhesion points under all conditions tested. Its staining showed that both RT4-D6P2T and primary SC cultured on aligned fibers had elongated actin fibers compared to the control condition and random fibers (Figure 2A). Finally, cells had an elongated morphology with their longitudinal axis parallel to the direction of the aligned gelatin nano-fibers (Figure 2A).

Bottom Line: Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers.B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers.The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043, Italy. sara.gnavi@unito.it.

ABSTRACT
Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

No MeSH data available.


Related in: MedlinePlus