Introduction: Preparation of 3D micropatterned porous scaffolds remains a great challenge for engineering of highly organized tissues such as skeletal muscle tissue. 2D micropatterned surfaces with periodic features are commonly used to guide the alignment of muscle myoblasts and myotubes and lead to formation of pre-patterned cell sheets. However, cell sheets from 2D patterned surfaces have limited thickness, and harvesting the cell sheets for implantation is inconvenient and can lead to less alignment of myotubes. 3D micropatterned scaffolds that guide cell alignment and tissue formation are desirable to engineer the organized skeletal muscle tissue. In this study, we developed 3D collagen scaffolds with concave microgrooves that mimic muscle basement membrane to engineer skeletal muscle tissue.
Materials and Methods: Parallel lines of frozen ice were prepared by dispensing pure water onto a copper plate and served as a template to prepare the microgrooved scaffolds. Cooled collagen aqueous solution was cast onto the frozen ice lines and covered with a glass plate. The whole construct was frozen in liquid nitrogen and freeze-dried to get a 3D microgrooved scaffold. Different scaffolds (G120, G200 and G380) were prepared by using frozen lines of different widths (120 ± 15, 200 ± 18 and 380 ± 22 μm). The pore structures were observed with a scanning electron microscope. Rat L6 skeletal muscle myoblasts were cultured in the microgrooved scaffolds in fusion medium. After 7 or 14 days of culture, cell-scaffold samples were fixed by 4% paraformaldehyde, rinsed in PBS and incubated in 0.2% Triton X-100. The samples were stained for F-actin, myosin heavy chain (MHC) and cell nuclei and observed with a fluorescence microscope (Olympus) or a confocal microscope.
Results and Discussion: The micropatterned scaffolds had aligned concave microgrooves that exhibited a semicircular shape in cross-section, which was inherited from the frozen ice line replicates. Rat L6 skeletal myoblasts were seeded at different seeding concentrations (0.4, 2.0, 4.0×106 cells/ml) onto the G200 scaffolds to investigate the influence of cell seeding density. Myoblasts adhered on the microgrooves, contracted into multi-cellular bundles in microgrooves. Cells seeded at an intermediate concentration (2.0×106 cells/ml) showed the best formation of aligned cell bundles. Furthermore, myoblasts were seeded onto the G120, G200, G380 scaffolds at the intermediate concentration (2.0×106 cells/ml) to investigate the effect of microgroove width. Scaffolds with wide microgrooves (G200 and G380) enabled formation of discrete and parellel cell bundles. Scaffolds with narrow microgrooves (G120) resulted in formation of some cell bundles in microgrooves and mostly cellular flakes covering most of the area of scaffolds. MHC staining showed that well aligned myotubes were formed within cell bundles in G200 and G380, while in G120 some myotubes were aligned in microgrooves and other myotubes possibly in cellular flakes had random orientation.
Conclusion: 3D collagen scaffolds with concave microgrooves were prepared by using ice lines. Highly aligned and multi-layered muscle bundle tissues were engineered by culturing L6 myoblasts in the microgrooved scaffolds. The formation and alignment of cell bundles in microoved scaffolds were dependent on the size of microgrooves and cell seeding concentration.
This study was supported by World Premier International Research Center Initiative on Materials Nanoarchitectonics and KAKENHI Grant Number 15K12548 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.