3D Printed Graphene-PLA Scaffolds Promote Cell Alignment and Differentiation

Abstract

Traumas and chronic damages can hamper the regenerative power of nervous, muscle, and connective tissues. Tissue engineering approaches are promising therapeutic tools, aiming to develop reliable, reproducible, and economically affordable synthetic scaffolds which could provide sufficient biomimetic cues to promote the desired cell behaviour without triggering graft rejection and transplant failure. Here, we used 3D-printing to develop 3D-printed scaffolds based on either PLA or graphene@PLA with a defined pattern. Multiple regeneration strategies require a specific orientation of implanted and recruited cells to perform their function correctly. We tested our scaffolds with induced pluripotent stem cells (iPSC), neuronal-like cells, immortalised fibroblasts and myoblasts. Our results demonstrated that the specific "lines and ridges" 100 µm-scaffold topography is sufficient to promote myoblast and fibroblast cell alignment and orient neurites along with the scaffolds line pattern. Conversely, graphene is critical to promote cells differentiation, as seen by the iPSC commitment to neuroectoderm, and myoblast fusions into multinuclear myotubes achieved by the 100 µm scaffolds containing graphene. This work shows the development of a reliable and economical 3D-printed scaffold with the potential of being used in multiple tissue engineering applications and elucidates how scaffold micro-topography and graphene properties synergistically control cell differentiation.

Keywords: 3D printing; G+; Grafylon; PLA; graphene; neuronal differentiation; regenerative medicine; scaffold; stem cells.

Figure 1

Frontal, lateral and axonometric views of scaffold design. (A) 100 µm series (B) 400 µm series. Scalebar of the fourth image of each panel is 100 µm and 400 µm, respectively.

Figure 2

Effects of bare Poly-L-lactic acid (PLA) or graphene@PLA (Gr@PLA) on SH-SY5Y. (A) Cell viability at either 24 or 72 h after cell seeding. Lower values at 24h are due to post-detachment stress. (B) Cell proliferation expressed as ratio between number of cells at 72h and 24h after seeding. Scaffolds do not significantly interfere with cell proliferation. All data represent the mean ± SD of at least three independent experiments. Statistical significance was determined with one-way ANOVA with Tukey’s correction.

Figure 3

Neuroectodermal commitment of iPSCs grown onto 3D-printed scaffolds. (A) PAX6 and (B) Nestin expression increases when cells are cultured on graphene@PLA (Gr@PLA) scaffolds. All data represents the mean ± SD of at least three independent experiments. Statistical significance was determined with a two tailed t-test. Significance at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) between samples is reported. CTRL condition consists of cells seeded onto common 24-well plates.

Figure 4

Alignment of SH-SY5Y cells to scaffold patterning. Values are at (A) day 1 and (B) day 6 from cell seeding. Cell orientation is influenced by scaffold patterning independently of its composition. CTRL condition consists of cells seeded onto common 24-well plates. All data represents the mean ± SD of at least three independent experiments. Statistical significance was determined with two-way ANOVA with Dunnet’s correction; significance at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) between samples is reported, (C–L) representative fields of cells grown onto scaffolds. (C) Day 1 control, (D) Day 1 PLA_100 µm, (E) Day 1 PLA 400 µm, (F) Day 1 Gr@PLA_100 µm, (G) Day 1 Gr@PLA_400 µm, (H) Day 6 control, (I) Day 6 PLA_100 µm, (J) Day 6 PLA 400 µm, (K) Day 6 Gr@PLA_100 µm, (L) Day 6 Gr@PLA_400 µm. Scalebar 50 µm.

Figure 5

Alignment of hTERT-immortalised fibroblasts to scaffold patterning. Values are at (A) day 1 and (B) day 6 from cell seeding. Cell growth is influenced by scaffold patterning independently of its composition. CTRL condition consists of cells seeded onto common 24-well plates. All data represent the mean ± SD of at least three independent experiments. Statistical significance was determined with two-way ANOVA with Dunnet’s correction; significance at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) between samples is reported, (C–L) representative fields of cells grown onto scaffolds. (C) Day 1 control, (D) Day 1 PLA_100 µm, (E) Day 1 PLA 400 µm, (F) Day 1 Gr@PLA_100 µm, (G) Day 1 Gr@PLA_400 µm, (H) Day 6 control, (I) Day 6 PLA_100 µm, (J) Day 6 PLA 400 µm, (K) Day 6 Gr@PLA_100 µm, (L) Day 6 Gr@PLA_400 µm. Scalebar 50 µm.

Figure 6

Effects of 3D-printed scaffolds on myoblasts. (A) Myoblasts alignment to scaffold pattern is strongly influenced by scaffold dimensionality, however it is independent from scaffold composition. (B) Fusion index is strongly increased by the combined effects of graphene and patterning. CTRL condition consists of cells seeded onto common 24-well plates. All data represents the mean ± SD of at least three independent experiments. Statistical significance of panel (A) with two-way ANOVA with Dunnet’s correction, whereas that of panel (B) was determined with one-way ANOVA with Tukey’s correction; significance at p < 0.05 (*), p < 0.01 (**), p < 0.001(***) and p < 0.0001 (****) between samples is reported, (C–G) representative fields of cells grown onto scaffolds. (C) control, (D) PLA_100 µm, (E) PLA 400 µm, (F) Gr@PLA_100 µm, (G) Gr@PLA_400 µm.