Chemotactic TEG3 Cells' Guiding Platforms Based on PLA Fibers Functionalized With the SDF-1α/CXCL12 Chemokine for Neural Regeneration Therapy

Abstract

(Following spinal cord injury, olfactory ensheathing cell (OEC) transplantation is a promising therapeutic approach in promoting functional improvement. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical concentration differences. Here we compare the attachment, morphology, and directionality of an OEC-derived cell line, TEG3 cells, seeded on functionalized nanoscale meshes of Poly(l/dl-lactic acid; PLA) nanofibers. The size of the nanofibers has a strong effect on TEG3 cell adhesion and migration, with the PLA nanofibers having a 950 nm diameter being the ones that show the best results. TEG3 cells are capable of adopting a bipolar morphology on 950 nm fiber surfaces, as well as a highly dynamic behavior in migratory terms. Finally, we observe that functionalized nanofibers, with a chemical concentration increment of SDF-1α/CXCL12, strongly enhance the migratory characteristics of TEG3 cells over inhibitory substrates.

Keywords: CXCL12; PLA nanofibers; SDF-1alpha; cell migration; electrospinning; gradients; olfactory ensheathing cells.

FIGURE 1

TEG3 cytoskeletal dynamics and phenotype morphology. (A) Lentiviral transfected LifeAct-eGFP TEG3 showing a high expression of membrane protrusions through the presence of motile lamellipodia; trailing process (white asterisk) and in the cell body (red asterisk); (B) intracellular distribution of F-actin and focal adhesions (FAs) on laminin substrate; (C) stain of integrin αvβ3; (D) flattened TEG3 cells showing membrane protrusion on one or both sides; and (E) FESEM image of a mix of bipolar (Schwann-type OECs, red arrows) and flattened (astrocyte-type OECs, white arrows) cells under in vitro conditions. Scale bars (A–C) – 10 μm; (D) – 10 μm; and (E) – 50 μm.

FIGURE 2

Cytomorphometric analysis of TEG3 over different diameter PLA nanofibers. Cells adhered onto fibers on coverslips with associated laminin of an averaged fiber diameter of (A) 350 nm, (B) 700 nm, and (C) 950 nm; (D) DAPI/phalloidin fluorescent image of cells adhered to a surface with (right) and without (left) 950 nm-fibers over an inhibiting coating of CSPG; (E) histogram showing the percentage of attachment of the cells with the different diameters of PLA nanofibers (biomaterial; ***p-value < 0.0001; **p-values 0,0017 and 0.0020; *p-value 0.0411; and N = 5); and (F) their shape circularity index of cells attached to the different fibers assessed in this work (***p-value < 0.0002; **p-value 0.0019; *p-value 0.0170; and N > 25). Scale bars (A) (same for B,C) – 50 μm; and (D) – 300 μm.

FIGURE 3

FESEM analysis of TEG3 over PLA nanofibers of differing diameters. FESEM images of (A) pure nanofibers of an average size of 950 nm aligned on coverslips with a laminin coating; (B, C) analysis of the cell morphology on the fibrous substrates showing that cell bodies are elongated along the axis of the fiber and extended trailing processes that are guided by fiber directionality; (D) peripheral lamellipodial waves bearing fine filopodia showing cell-cell contact and cell group migration over the nanofibers; (E) cell membrane on the migratory front interacting with the nanotopography of PLA nanofiber (asterisk); (F) detail of the elongation of eGFP-TEG3 over a stained fiber (red) with immunofluorescence techniques. Scale bars: (A) – 1 μm., (B) (same for (C)) – 50 μm; (D) – 5 μm; (E) – 7.5 μm; and (F) – 10 μm.

FIGURE 4

Enhanced adhesion and migration of TEG on SDF-1α functionalized nanofibers. Scheme showing how the paraffin frames with the fibers on the top were dipped into the different aqueous solutions for activation and functionalization (A); optical microscope images showing the maintained structure of the coated fibers in bright-field (B), the homogeneous functionalization of the nanofibers with the chemotactic agent SDF-1α/CXCL12 by linking fluorescent SDF-1α antibodies (C), and (D) the concentration of TEG3 cells in two areas with different concentrations of the chemotactic SDF-1α/CXCL12; (E) detailed instrumental scheme of the fibers within the paraffin frame laid onto a glass cover-slide coated with inhibitory CSPG where the cells are seeded using a PDMS template container and removed after confluence to let the cells migrate; (F,G) immunofluorescence images of the culture on both sides of the initial cell deposit at a time-point of 6 days showing the differences in number and distance migrated (white arrows); and (H) quantification of the covered distance by the cells at 4 and 6 days. Kruskal–Wallis test. ***p-value 0,0006; **p-value 0,0026. Scale bars (C,D) – 150 μm and (F,G) 300 μm.