Skeletal muscle tissue engineering: methods to form skeletal myotubes and their applications

Abstract

Skeletal muscle tissue engineering (SMTE) aims to repair or regenerate defective skeletal muscle tissue lost by traumatic injury, tumor ablation, or muscular disease. However, two decades after the introduction of SMTE, the engineering of functional skeletal muscle in the laboratory still remains a great challenge, and numerous techniques for growing functional muscle tissues are constantly being developed. This article reviews the recent findings regarding the methodology and various technical aspects of SMTE, including cell alignment and differentiation. We describe the structure and organization of muscle and discuss the methods for myoblast alignment cultured in vitro. To better understand muscle formation and to enhance the engineering of skeletal muscle, we also address the molecular basics of myogenesis and discuss different methods to induce myoblast differentiation into myotubes. We then provide an overview of different coculture systems involving skeletal muscle cells, and highlight major applications of engineered skeletal muscle tissues. Finally, potential challenges and future research directions for SMTE are outlined.

Figures

FIG. 1.

Schematic illustration of the concept of skeletal muscle tissue engineering showing three different types of culture techniques for generating muscle tissue. Color images available online at www.liebertpub.com/teb

FIG. 2.

Anatomy of a skeletal muscle and a sarcomere. (A) From SEER training on structure of skeletal muscle, U.S. National Institute of Health, National Cancer Institute (12 July 2012). http://training.seer.cancer.gov/anatomy/muscular/structure.html(B) Micrograph of a sarcomere adapted with permission from Sosa et al. Copyright © 1994, Elsevier, and schematic. Color images available online at www.liebertpub.com/teb

FIG. 3.

C2C12 cells cultured on an array of large fibers. (A) Thirty minutes after seeding. (B) Gaps between fibers were closed after 5 weeks of culture and a cell sheet was formed. (C) After 10 weeks in culture, myotubes were striated (DIC image). (D) Cross-section of the muscular tissue fixed and stained by hematoxylin and eosin. The fiber plane is in the upper section (scale bars for [A–D] 50 μm). Reprinted with permission from Neumann et al. Copyright © 2003, Mary Ann Liebert, Inc.

FIG. 4.

Fiber-fabrication procedure by Polystyrene Spinneret-based Tunable Engineered Parameter (STEP). (A) Schematic showing the building of fibers by STEP. (B) STEP fiber types that can be fabricated by one set of fibers running in a parallel manner (left), two sets of fibers running perpendicular to each other (middle), and one set of fibers running in a parallel manner with a hollowed-out support base (right). (C) SEM and TEM pictures used to quantify the STEP fiber diameter and length. (D) SEM image showing the cell attachment to the polystyrene STEP fibers. (E) Images in fluorescence of polystyrene STEP fibers coated with Alexa649-conjugated fibrin (right) and uncoated (left). (F) Polystyrene STEP fibers can be printed on by inkjet printing. (Scale bars: B, 2 mm; C, 100 μm and right photo 2 μm; D, 100 μm; E, 200 μm). Reprinted with permission from Ker et al. Copyright © 2011, Elsevier.

FIG. 5.

Oriented cellulose nanowhiskers of 10–15-nm diameter were produced by spin-coating. The response of myoblasts to the surfaces was assessed by atomic force microscopy 12 h after seeding. The inset picture shows a large-scale image of the whole cell (inset scale bar=20 μm), whereas the yellow arrow indicates the direction of the nanowiskers. The main image shows a closeup scan of the area bounded by the dashed box in the inset picture (scale bar=5 μm), whereas the white arrows indicate filopodia. Reprinted with permission from Dugan et al. Copyright © 2010, American Chemical Society.

FIG. 6.

(a) A Silicon wafer coated with SU-8 was patterned by UV through (b) a photomask (scale bars=2 mm; inset=500 μm). (c) Optical profile of the master mold. (d) Polydimethylsiloxane (PDMS)-negative replica (scale bars=1 mm; inset vertical cross-section 500 μm). (e) PDMS-positive replica (scale bars=1 mm; inset vertical cross-section 500 μm). (f) Cells in hydrogel prepolymer solution were poured in the PDMS mold and incubated at 37°C to allow (g) hydrogel polymerization. (h) The culture medium was then added and the cells were cultured for 2 weeks. The hydrogel was fixed on a Velcro frame (scale bars in f–h 5 mm). Reprinted with permission from Bian et al. Copyright © 2009, Nature Publishing Group. Color images available online at www.liebertpub.com/teb

FIG. 7.

3T3 fibroblasts encapsulated in 5% GelMA hydrogels patterned into rectangular microconstructs [50 μm (w)×800 μm (l)×150 μm (h) with 200-μm interlines]. (A) Hydrogel stained with Rhodamine B showing initial microconstruct at day 0 and phase-contrast images of cell-laden microconstructs at days 1, 4, and 7 of culture. The red arrows indicate points of contact between neighboring lines at day 4 of culture, with tissue convergence at day 7 of culture. (B) Three-dimensional (3D) tissue construct (1×1 cm2) at day 7 of culture. (C) F-Actin staining showing the middle of the 3D tissue construct with aligned actin fibers. Reprinted with permission from Aubin et al. Copyright © 2010, Elsevier. Color images available online at www.liebertpub.com/teb

FIG. 8.

Micropatterned substrate building process and cell culture. (A) Schematic showing the preparation of the micropatterned substrate. (B) Phase-contrast images of the different micropatterns with cells: lines of different widths (300 μm, 150 μm, 80 μm, 40 μm, 20 μm, and 10 μm), tori of different inner diameters (40 μm, 100 μm, and 200 μm), and hybrid patterns of different arc degrees (30°, 60°, and 90°), (scale bar=100 μm). Reprinted with permission from Bajaj et al. Copyright © 2011, Royal Society of Chemistry. Color images available online at www.liebertpub.com/teb

FIG. 9.

Schematic showing the micropatterning of myotubes by the use of a thin PDMS stencil membrane. A BSA-coated membrane was attached on the surface of a Petri dish and C2C12 were seeded on the membrane. After culturing in differentiation medium, the membrane was peeled off under a microscope to free the micropatterned myotubes, which can be removed. Reprinted with permission from Shimizu et al. Copyright © 2010, Elsevier. Color images available online at www.liebertpub.com/teb

FIG. 10.

Micropatterning of heterotypic cell sheets. (a) Patterning of an SU-8 layer spin coated on an indium tin oxide (ITO) electrode by UV through a photomask. (b) After development, SU-8 micropatterns are formed on the ITO electrode. (c) The substrate is then coated with a weak cell-adhesive polyelectrolyte and (d) subjected to electrochemical polarization, which induced the dissolution of the polyelectrolyte only from the ITO regions. (e) The ITO regions are backfilled with a cell-repellent polymer PLL-g-PEG and (f) a first cell type is seeded whereas the nonadherent cells are washed away. (g) The PLL-g-PEG monolayer is then removed by a second electrochemical step. (h) The ITO regions are backfilled with PLL-g-PEG/PEG-RGD, which is a cell-adhesive monolayer. (i) The second cell type is seeded and the nonadherent cells are washed away. (j) After 1 day of culture, the PLL-g-PEG/PEG-RGD monolayer is dissolved by a final electrochemical step, which allows the whole cell sheet to detach. Indeed, due to their weak interaction with the substrate, the cells on weak cell-adhesive regions also detach easily. PEG, poly(ethylene glycol). Reprinted with permission from Guillaume-Gentil et al. Copyright © 2010, Springer Science+Business Media, LLC. Color images available online at www.liebertpub.com/teb

FIG. 11.

Patterning of two different cell populations by dielectrophoresis (DEP). (A) An interdigitated array of four-electrode subunit was used to pattern cells. (B) By applying an AC voltage, the n-DEP force was induced and cells were directed toward weaker region of electric field strength. (C) Cells in excess were removed. (D) A second cell type was loaded into the device and guided to other areas by changing the AC voltage mode. Reprinted with permission from Suzuki et al. Copyright © 2008, Elsevier. Color images available online at www.liebertpub.com/teb

FIG. 12.

Effects of electrical stimulation on muscular protein and sarcomere development. C2C12 myotubes were stimulated 24 h with electrical pulses (40 V, 1 Hz, 2 ms duration). The cell lysates were analyzed by western blot (left) and cells were fixed (right) and stained with DAPI for nucleus (blue), anti-sarcomeric α-actinin antibody for sarcomeric α-actinin (red), and phalloidin for MHC (green). Reprinted with permission from Nedachi et al. Copyright © 2008, The American Physiological Society. Color images available online at www.liebertpub.com/teb

FIG. 13.

Topographical effects on C2C12 differentiation and myotube formation at different days in differentiation culture medium. Cells were cultured on unpatterned (control) and patterned substrates with different shapes (scale bar=100 μm) and stained with DAPI for nucleus (blue) and anti-MHC (green). Reprinted with permission from Bajaj et al. Copyright © 2011, Royal Society of Chemistry. Color images available online at www.liebertpub.com/teb

FIG. 14.

Stiffness effects on myotube striation at 4 weeks in differentiation culture medium. C2C12 were cultured on collagen-patterned substrates with different stiffness (scale bar=20 μm) and stained with DAPI for nucleus (blue) and anti-MHC (green). Myosin striation occurred only when cells were cultured on intermediated stiffness substrate. Reprinted with permission from Engler et al. Copyright © 2004, Rockefeller University Press. Color images available online at www.liebertpub.com/teb

FIG. 15.

Prevascularization of five-layer myoblast sheet constructs in vitro. (A) Schematic showing the construct made of human umbilical vein endothelial cells (HUVECs) sandwiched between myoblast cell layers. (B) HUVECs in the construct were stained with anti-human CD31 antibody (green, upper photo) or UEA-I (red, lower photo) whereas nuclei were stained with Hoechst 33342 (blue). HUVECs networked through the cell layers and formed capillary-like structures (white arrowheads) at day 4 of coculture. Asterisk shows the position of the fibrin gel used as substrate. Reprinted with permission from Sasagawa et al. Copyright © 2010, Elsevier. Color images available online at www.liebertpub.com/teb

FIG. 16.

SEM image of a rodent neuromuscular junction. Components are as follows: nerve terminal (N), muscle fiber (M), Schawnn cell (SC#), round synaptic vesicles containing ACh docked in the active zone of the nerve terminal (*), and synaptic cleft with basal lamina (SBL). Reprinted with permission from Wu et al. Copyright © 2010, The Company of Biologists Ltd.

FIG. 17.

Engineering of miniature bioartificial muscles (mBAMs) on PDMS microposts in a 96-well plate for drug screening. PDMS microposts are 7-mm high, 7 mm Ø, and 4-mm apart. The mBAM shown in the well is 5 days postcasting whereas at day 7 the mBAM showed aligned myofibers after staining for sarcomeric tropomyosin. Reprinted with permission from Vandenburgh. Copyright © 2010, Mary Ann Liebert, Inc.

FIG. 18.

Comparison of energy, greenhouse gas (GHG) emissions, and land and water used between different sources of meat production. Reprinted with permission from Tuomisto and Teixeira de Mattos. Copyright © 2011, American Chemical Society. Color images available online at www.liebertpub.com/teb