Helical nanofiber yarn enabling highly stretchable engineered microtissue

Abstract

Development of microtissues that possess mechanical properties mimicking those of native stretchable tissues, such as muscle and tendon, is in high demand for tissue engineering and regenerative medicine. However, regardless of the significant advances in synthetic biomaterials, it remains challenging to fabricate living microtissue with high stretchability because application of large strains to microtissues can damage the cells by rupturing their structures. Inspired by the hierarchical helical structure of native fibrous tissues and its behavior of nonaffine deformation, we develop a highly stretchable and tough microtissue fiber made up of a hierarchical helix yarn scaffold, scaling from nanometers to millimeters, that can overcome this limitation. This microtissue can be stretched up to 15 times its initial length and has a toughness of 57 GJ m^-3 More importantly, cells grown on this scaffold maintain high viability, even under severe cyclic strains (up to 600%) that can be attributed to the nonaffine deformation under large strains, mimicking native biopolymer scaffolds. Furthermore, as proof of principle, we demonstrate that the nanotopography of the helical nanofiber yarn is able to induce cytoskeletal alignment and nuclear elongation, which promote myogenic differentiation of mesenchymal stem cells by triggering nuclear translocation of transcriptional coactivator with PDZ-binding motif (TAZ). The highly stretchable microtissues we develop here will facilitate a variety of tissue engineering applications and the development of engineered living systems.

Keywords: bioinspired scaffold; muscle regeneration; myogenesis; nanofiber yarn; stretchable tissue.

Fig. 1.

Fabrication and characterization of helix microtissue fiber. (A) Schematic illustration of the structure of hierarchical helix nanofiber yarn. (B and C) Scanning electron micrographs of an unstrained (B) and a strained (C) hierarchical helix nanofiber yarn. (D) Stress–strain curve of helix nanofiber yarn shows that the helix scaffold can be stretched up to 15 times its initial length under a stress more than 80 MPa. (E) Quantifications of fracture strain, fracture strength, and fracture toughness show that the helix scaffold’s toughness is 6 times larger than the primary yarn by elevating fracture strain rather than facture strength. ***P < 0.001. n.s., not significant. (F and G) Image (F) and quantification (G) of LIVE/DEAD staining of seeded MEFs show that three different types of cells maintain high cell viability on the helix scaffold during 5 d of culturing.

Fig. 2.

Hierarchical helix scaffold shelters cells from large cyclic straining and bending. (A) Schematic illustration of cycling strain of a hierarchical helix scaffold. (B) LIVE/DEAD staining of seeded cells under cycling strains. The results show that cells maintain a consistently high viability despite different strain ratios. (C) Seeded cells maintain high viability on the helix scaffold under different strains up to 600%. As a comparison, the cell viability dramatically decreases as the strain increases when cells are cultured on the primary yarn. (D) Cells on the helix scaffold maintain a spreading morphology and remain attached (>83%) under 600% cycling strain, while most cells (65%) on the primary yarn round up and detach under 50% cyclic strain. (E) Cells on the helix scaffold maintain high viability at different strain rates ranging from 1/32 to 16 min⁻¹, while cells on the primary yarn round up and detach under 50% cyclic strain. (F) Schematic illustration of cycling bending of a hierarchical helix scaffold. (G) Cells on the helix scaffold maintain high viability under different bend ratios up to 270°. In comparison, cell viability decreases as the bend ratio is larger than 150° when cells are cultured on the primary yarn. (H) Cells on the helix scaffold maintain a spreading morphology and remain attached (>95%) under different bend ratios up to 270°, while cells on the primary fiber round up and detach.

Fig. 3.

Theoretical modeling of straining the hierarchical helix scaffold. (A) Simulated results show the local strain of the hierarchical helix scaffold under an applied 50% engineering strain, showing that the local material strain is much smaller than the applied engineering strain. (B) The field of local material strains on the helix scaffold and primary yarn under the same engineering strains, showing a much larger local material strain on the primary yarn compared with the helix scaffold. (C) The simulated correlations between local strain and engineering strain show that the local strains on the helix scaffold are much smaller than those on the primary yarn under the same engineering strain. Experimental measurements of cell elongation also fit well with the simulated local material strain of the helix scaffold. (D) Local orientations of seeded cells on the helix scaffolds are consistent with simulated local orientations of nanofibers under the same engineering strain.

Fig. 4.

The helix scaffold promotes myogenic differentiation of MSCs via altering physical properties of cells and translocating TAZ. (A–C) Images (A) and quantification of myosin heavy chain (MHC) staining ratio (B) and intensity (C) show that the helix scaffold promotes myogenic differentiation of MSCs. (D–F) Fluorescent images (D) and quantifications show that the helix scaffold elongates the cells to a larger aspect ratio (E) and reduces the cell volume (F) compared with those cultured on a 2D substrate. (G–I) Fluorescent images (G) and quantifications show that the helix scaffold elongates the nucleus to a larger aspect ratio (H) and reduces the nuclear volume (I) compared with those culture on a 2D substrate. (J–L) Fluorescent images (J) and quantifications show that the helix scaffold promotes TAZ nuclear translocation compared with 2D substrate (K and L). (M–O) Overexpression of TAZ in MSCs leads to a comparable myogenesis ratio in cells cultured on the helix scaffold and a 2D substrate. (P and Q) Helix scaffold guarantees a high viability and an elevated myogenic differentiation ratio of MSCs under cycling 600% strains. As a comparison, only a small portion of MSCs on the primary yarn maintains health and goes through myogenic process. *P < 0.05, **P < 0.01. n.s., not significant; overE, overexpression; Pri-, primary.