Novel ultrashort self-assembling peptide bioinks for 3D culture of muscle myoblast cells

Abstract

The ability of skeletal muscle to self-repair after a traumatic injury, tumor ablation, or muscular disease is slow and limited, and the capacity of skeletal muscle to self-regenerate declines steeply with age. Tissue engineering of functional skeletal muscle using 3D bioprinting technology is promising for creating tissue constructs that repair and promote regeneration of damaged tissue. Hydrogel scaffolds used as biomaterials for skeletal muscle tissue engineering can provide chemical, physical and mechanical cues to the cells in three dimensions thus promoting regeneration. Herein, we have developed two synthetically designed novel tetramer peptide biomaterials. These peptides are self-assembling into a nanofibrous 3D network, entrapping 99.9% water and mimicking the native collagen of an extracellular matrix. Different biocompatibility assays including MTT, 3D cell viability assay, cytotoxicity assay and live-dead assay confirm the biocompatibility of these peptide hydrogels for mouse myoblast cells (C2C12). Immunofluorescence analysis of cell-laden hydrogels revealed that the proliferation of C2C12 cells was well-aligned in the peptide hydrogels compared to the alginategelatin control. These results indicate that these peptide hydrogels are suitable for skeletal muscle tissue engineering. Finally, we tested the printability of the peptide bioinks using a commercially available 3D bioprinter. The ability to print these hydrogels will enable future development of 3D bioprinted scaffolds containing skeletal muscle myoblasts for tissue engineering applications.

Keywords: 3D cell culture; 3D scaffold; bioinks; biomaterials; skeletal muscle cells; tissue engineering.

Figure 1

Ultrashort peptides self-assemble into three-dimensional nanofibrous networks. Field emission scanning electron microscopy images of 2.5 mg/mL bovine collage type I (A, B, C), 4 mg/mL CH-01 (D, E, F) and 3 mg/mL CH-02 (G, H, I).

Figure 2

Graphical representation of MTT assay of mouse myoblast cells incubated with different peptide concentrations for 24 h. Alginate-Gelatin (1:1) was used as positive control (A), CH-01(C), CH-02 (D) and a standard curve for a known number.

Figure 3

Representation of cytotoxicity on mouse myoblast cells after 24 h incubation with different peptide concentrations. Alginate- Gelatin used as positive control (A), CH-01 (B), and CH-02 (C). Error bars, mean ± S.D.

Figure 4

3D viability assay of mouse myoblast cells encapsulated in peptide hydrogels, 4 mg/mL CH-01 and 3 mg/mL and 30 mg/mL alginate-gelatin (1:1).

Figure 5

Live/dead staining of mouse myoblast cells encapsulated in peptide hydrogels, 4 mg/mL CH-01 and 3 mg/mL CH-02, and 30 mg/mL alginate-gelatin (1:1), for different time points. Alginate-Gelatin used as positive control (B, E, H), CH-01 (C, F, I), and CH-02 (D, G, J) at day 2, 4 and 8, respectively. Scale bars 100 μm.

Figure 6

Overlaid confocal fluorescence images of mouse myoblast cells encapsulated in peptide (4 mg/mL CH-01 and 3 mg/mL; CH- 02) and alginate-gelatin (1:1) hydrogels. The encapsulated cells were cultured for different days and finally analyzed using fluorescence confocal microscopy (Nucleus shown in blue, F-actin shown in red and vinculin in green). (A) Mouse myoblast cells cultured on tissue culture plate (TCP). Alginate-gelatin (B, C, D), CH-01 (E, F, G), and CH-02 (H, I, J) at day 2, 4, and 8, respectively. Scale bar is 20 μm.

Figure 7

Quantification of C2C12 alignment within different scaffolds for different time points. Column 1 shows the fluorescent image of the Myoblast cells within different scaffold; Alginate/gelatin (A, D, G), CH-01 (B, E, H) and CH-02 (C, F, I) at day 2, 4 and 8, respectively. Colum 2 shows the grayscale images to perform FFT analysis. Column 3 shows the two-dimensional FFT of the grayscale images image. The bottom graphs show the FFT plot for CH-01, CH-02 and alginate-gelatin which confirm the alignment of myoblast cells after 2, 4, and 8 days, (scale bars = 20 μm).

Figure 8

Bioprinting of peptide hydrogels; Bioprinted peptide hydrogel in different structures: (A) Circle; (B) Square.