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Topographic control has been widely used to facilitate the process of cell fusion in skeletal muscle formation. Muscle tubes that deviate from the groove boundary generally have a consistent chiral orientation, an issue that has long been a concern. Recently, the team of Prof. Ting-Hsuan Chen and Professor Miu Ling Lam from the City University of Hong Kong conducted research on the formation of scalable models of skeletal muscle canals through synergistic cell micromorphology and chiral nematics. The research results were published in Biofabrication on March 10, 2023 under the title "Scalable pattern formation of skeletal myotubes by synergizing microtopographic cues and chiral nematics of cells".
A scalable and controlled method is designed to guide skeletal muscle tube formation. By inducing C2C12 myocytes onto groove patterns of different widths, enhanced chiral orientation of cells developed on wide grooves was observed from the first day of induction. Active chiral movements involving cell migration and chiral rotation of the nucleus subsequently lead to uniform chiral orientation of the myotube. These chiral tube formations have properties that enhance length, diameter, and wide groove contractility. Latrunculin A (Lat A) treatment inhibits chiral rotation and migration of cells and myotube formation, suggesting that chiral centripetal nature of cells is essential for myotube formation. Finally, by arranging a wide groove/stripe pattern with corresponding compensation angles to synergize the microtopography and the chiral centripetality of cells, a complex and scalable myotube pattern is formed, which provides a new strategy for skeletal muscle tissue engineering.
Figure 1 Analysis of myotubes formed on grooved pattern RGD-Au-NOA chips
Myotube formation was explored by inducing myocyte on RGD-AU-NOA matrices with nano- or microgroove patterns of different widths. Stained with α-actin 1 8 days after differentiation, examination analysis in a fixed area showed that the myotube density on the narrow groove was 15%-25% less than in the planar control group and 10% more on the 100 μm trough. The density of myotubes detected on 50um and 200um grooves is comparable to that of planar controls. In addition, the mean myotube length of all width groove patterns increased significantly compared to the planar control. This improvement may be due to the fact that the groove pattern provides topographic factors that guide the cell arrangement, promoting end-to-end cell contact and confluency rates. In addition, the chiral orientation of the myotube is also very evident in the wide groove pattern, and the directional angle of the myotube is defined as the long axis of the myotube relative to the groove/chip boundary. The orientation of the myotube is randomly distributed on a flat base, but is uniaxial on all groove patterns. The myotubes are more arranged along grooves with a width of 0.4 and 2 μm horizontally. However, significant negative deviations in orientation angles were found on grooves 50, 100 and 200 μm wide, demonstrating a change in chiral orientation accompanied by the formation of myotubes.
In summary, the wide groove (100 μm) pattern can induce myocyte differentiation into mature myotubes with longer length, larger diameter, and better contractility compared to ultrafine grooves (0.4 and 2 μm), indicating significantly enhanced myotube production on wide grooves compared to myotubes on ultrafine grooves. Chiral orientation also appears during the formation of longer and denser myotubes, suggesting that cellular chirality is highly involved in the myogenesis process.
Figure 2 Cell orientation before and at the beginning of differentiation
In order to better understand the effect of groove width on myotube formation, the orientation, rotation and migration of cells on grooved RGD-Au-NOA chips were further studied. For measurements of cell orientation, the results show that cells are highly arranged at the edge of the groove at 0.4 and 2 μm. In contrast, most cells are randomly oriented on wide grooves (50, 100, and 200 μm) seven h after cell seeding. After that, as the cells converge, a negative hemichial arrangement appears. Indicates that chiral orientation appeared early before the formation of the myotube matured. To understand the development of chiral sexual orientation, cell migration on Day 1 in GM and Day 2 in DM was tracked. The migration trajectory of muscle cells on the groove pattern is first monitored. On grooves of 0.4 and 2 μm, cells rapidly sense the groove microtopography and migrate along the groove edge within two days. On day 1 of GM, cells migrate in all directions on grooves of 50, 100, and 200 μm. Illustrating the additional space between cells on low-density topographic factors, allowing cells to migrate in different directions, indicating that chiral orientation formation is based on active centripetal nature of cells, i.e., propagation from cells at the edge of the groove to distant cells as they approach confluence. To explore the chiral motility of cells, we further investigated the nuclear rotation and migration angle of cells migrating on narrow groove (2 μm) and wide groove (100 μm) during migration. The results showed that the nucleus rotated unbiased on the 2 μm groove, while the nucleus showed ACW-biased rotation on the 100 μm groove for both days.
The above results show that during cell migration, the nucleus actively rotates, and the chirality within the cell may guide the establishment of chiral direction and cell migration, and ultimately lead to uniform chiral direction throughout the culture through active centripetal nature of the cell. Conversely, on narrow grooves, the chirality of nucleus rotation is inhibited, resulting in the loss of chiral orientation in groove suprapause of 0.4 and 2 μm. Since mature myotubes with longer length, diameter and better contractility were also found on wide grooves (100 μm) in the later stages of differentiation, it is suggested that the chirality of cells may be an indispensable and important element for strengthening muscle formation.
Fig. 3 Actin inhibitors treated plasma with bands, regulating cell specificity and myotube differentiation
To further elucidate the nature of actin, three actin inhibitors were screened using single-cell rotation assays.
Cells are scattered on fibronectin (FN)-coated ring islands surrounded by cell-repellent Pluronic coating. In rotation analysis, only single cell counts with round shapes. The results showed that muscle cells showed a significant ACW rotation deviation of 55.5%, NSC enhanced it to 62.08%, and Simfh 2 enhanced it to 57.01%. However, for treatment of Lat A, cells were reversed to a 56.4% CW bias. Thus, Lat A is used to study the effect of cell chirality on myotube formation. Next, the myogenic effect based on Lat A treatment was discussed. Myocytes are cultured on a non-adherent dish with plasma-treated microstrips with a width of 500 μm. The results showed that for the untreated control group, the arrangement of the myotube and its nuclei was random before induction, but a negative direction appeared after myotube formation, and after Lat A supplementation in DM, both the myotube and nucleus showed an opposite positive bias. With treatment with Lat A in DM, myotube formation is also reduced by about 10%. By simultaneously monitoring nucleus rotation and migration on day 2, it was found that in the control group, muscle cells maintained ACW-rotation bias, while under Lat A treatment, exhibited CW-rotation bias. At the same time, migration (including lateral motion) is also inhibited by Lat A. Thus, Lat A treatment inhibits the active chiral mitochondria of cells, ultimately leading to a decrease in myotube formation.
Fig. 4 Formation of myotube expandable mode
To expand the myotube to expandable marking, the myotube is arranged in large-scale concentric and radial shapes. Choose a 100 μm wide slot as the base unit. First, a circle with a radius of 1 cm is divided into 12 equal regions. In each sector area, the myotube is designed parallel to the outer tangent to form a concentric ring pattern and parallel to the line in each sector area to form a radial pattern. Then 100 μm grooves are arranged in each sector area, with a certain compensation angle to the desired direction of the mark. After 8 days of differentiation, concentric rings and radial patterns of the myotube were achieved by arranging grooves on the hydrogel. The possibility of incorporating the cell-free fraction using plasma-treated streaks along with the desired myotube formation arrangement is also explored here. Use 100 μm wide plasma-treated cell adhesion bands separated from 50 μm wide untreated bands with appropriate compensation angles. On this basis, a "CITYU" mode myotube can be formed on a 100 μm plasma-treated fringed (cell adhesion) and 50 μm untreated spatial (cell rejection) array, demonstrating that appropriate compensation angle is a successful engineering strategy to guide the myotube to form a scalable pattern.
In summary, microtopographic factors using microgrooves to guide myotube formation resulted in enhanced chiral orientation in wide grooves (50 and 100 μm) and enhanced length, diameter, and contractility compared to those on narrow grooves (0.4-2 μm). Studies of cell orientation, migration, and nuclear rotation on groove patterns have shown that active chiral centripetality of cells is key to promoting myotube differentiation. Since the chiral arrangement of the myotube is inevitable and a natural consequence of active chirality, a strategy was developed to use the compensation angle to synergize microtopographic factors and the chirality of cells to guide the formation of myotube pattern. Laying the foundation for designing complex, scalable patterns, the results will have a significant impact on pattern design in tissue reconstruction studies.
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