Graphene Oxide Scaffold Stimulates Differentiation and Proangiogenic Activities of Myogenic Progenitor Cells

Abstract

The physiological process of muscle regeneration is quite limited due to low satellite cell quantity and also the inability to regenerate and reconstruct niche tissue. The purpose of the study was to examine whether a graphene oxide scaffold is able to stimulate myogenic progenitor cell proliferation and the endocrine functions of differentiating cells, and therefore, their active participation in the construction of muscle tissue. Studies were carried out using mesenchymal cells taken from 6-day-old chicken embryos and human umbilical vein endothelial cells (HUVEC) were used to assess angiogenesis. The graphene scaffold was readily colonized by myogenic progenitor cells and the cells dissected from heart, brain, eye, and blood vessels did not avoid the scaffold. The scaffold strongly induced myogenic progenitor cell signaling pathways and simultaneously activated proangiogenic signaling pathways via exocrine vascular endothelial growth factor (VEGF) secretion. The present study revealed that the graphene oxide (GO) scaffold initiates the processes of muscle cell differentiation due to mechanical interaction with myogenic progenitor cell.

Keywords: angiogenesis; graphene oxide; myogenic progenitor cells.

Figure 1

Graphene oxide scaffold morphology. (A) Atomic force microscopy images and (B) a topography model of the surface of the graphene oxide scaffold on a polystyrene culture plate. (C) Transmission electron microscopy image of graphene oxide. (D) Atomic force microscopy images and (E) a topography model of the graphene oxide scaffold surface on a flat silicon wafer. (F) Scanning electron microscopy image of the graphene oxide scaffold.

Figure 2

Analysis of the cell localization and viability on the GO scaffold. (A) Viability and (B) morphologies of progenitor muscle cells from hind limb (PMC) on the GO scaffold. (C) Viability and (D) morphology of progenitor eye cells (PEC) on the GO scaffold. (E) Viability and (F) morphology of progenitor heart-derived cells (PHC) on the GO scaffold. (G) Viability and (H) morphology of cells derived from brain (progenitor nerve cells, PNC) on the GO scaffold. (I) Viability and (J) morphology of cells from a chorioallantoic membrane’s blood vessel (progenitor vessel cells, PVC) on the GO scaffold. Morphology was assessed on the edge of the GO scaffold via light microscopy with phase contrast and 200 × magnification. Statistical significance is indicated with different superscripts (unpaired t-test; p < 0.05). (K) Pattern of GO islands used in cell localization and morphology analysis. Acronyms: C, control; GO, graphene oxide; RV, relative value.

Figure 3

PMC morphology after a 96-h incubation and PMC migration on a GO scaffold. (A) Scanning electron microscope visualization of PMC on the edge and (B,C) the center of the GO scaffold. (D) Visualization of the whole GO scaffold overgrown by PMC. (E) Representative images of the migration analysis of PMC throughout the GO scaffold. Image of the initial (“0 h”) position of the PMC and their positions after an 18-h incubation period on a polystyrene culture plate (“C”) and a graphene oxide scaffold (“GO”). (F) Fold decreases of the cell-free areas measured as the difference determined between 0 h and 18 h images. Arrows indicate the edge of the GO scaffold. Statistical significance is indicated with different superscripts (unpaired t-test; p < 0.05). Abbreviations: C, control; GO, graphene oxide; RV, relative value.

Figure 4

Analysis of mRNA expression and protein level. (A) Vascular endothelial growth factor A (VEGF-A) protein level in the cell medium after incubation of PMC on a standard cell culture plate (control—“C”), PMC on the graphene oxide scaffold (“GO scaff”), PMC on a standard cell culture plate treated with GO (final concentration—10 μg/mL). Expression level of the genes coding (B) VEGF-A, (C) basic fibroblast growth factor (bFGF), (D) myoblast determination protein 1 MyoD, (E) ATP synthase F1 subunit beta ATP5B, (F) proliferating cell nuclear antigen PCNA. Statistical significance is indicated with different superscripts (one-way ANOVA; p < 0.05). Abbreviations: RU, relative units.

Figure 5

Analysis of angiogenic properties of PMC media. Images of human umbilical vein endothelial cells (HUVEC) tube formation in (A) the control medium and (B) the negative control medium with the post-incubation addition of the medium from the PMC cultured under standard conditions as the control, and (C) with the post-incubation addition of the medium from the PMC cultured on a graphene oxide scaffold. (D) Graph showing the mean number of junctions of HUVEC tubes in the field of view. Values are expressed as mean ± standard deviation. Statistical significance is indicated with different superscripts (one-way ANOVA; p < 0.05). (E) Implant from the peripheral blood vessel of a chicken embryo incubated in the medium derived from standard PMC cultures (negative control) and (F) analysis of cells incubated in the medium from the PMC cultured on graphene oxide. Abbreviations: RV, relative value; N, negative control; C, control; GO scaff, experimental group.