A Comparison of Ovine Facial and Limb Muscle as a Primary Cell Source for Engineered Skeletal Muscle

Abstract

Volumetric muscle loss (VML) contributes to the number of soft tissue injuries that necessitate reconstructive surgery, but treatment options are often limited by tissue availability and donor site morbidity. To combat these issues, our laboratory has developed scaffold-free tissue-engineered skeletal muscle units (SMUs) as a novel treatment for VML injuries. Recently, we have begun experiments addressing VML in facial muscle, and the optimal starting cell population for engineered skeletal muscle tissue for this application may not be cells derived from hindlimb muscles due to reported heterogeneity of cell populations. Thus, the purpose of this study was to compare SMUs fabricated from both craniofacial and hindlimb sources to determine which cell source is best suited for the engineering of skeletal muscle. Herein, we assessed the development, structure, and function of SMUs derived from four muscle sources, including two hindlimb muscles (i.e., soleus and semimembranosus [SM]) and two craniofacial muscles (i.e., zygomaticus major and masseter). Overall, the zygomaticus major exhibited the least efficient digestion, and SMUs fabricated from this muscle exhibited the least aligned myosin heavy chain staining and consequently, the lowest average force production. Conversely, the SM muscle exhibited the most efficient digestion and the highest number of myotubes/mm²; however, the SM, masseter, and soleus groups were roughly equivalent in terms of force production and histological structure. Impact Statement An empirical comparison of the development, structure, and function of engineered skeletal muscle tissue fabricated from different muscles, including both craniofacial and hindlimb sources, will not only provide insight into innate regenerative mechanisms of skeletal muscle but also will give our team and other researchers the information necessary to determine which cell sources are best suited for the skeletal muscle tissue engineering.

Keywords: cell inhomogeneity; muscle-derived progenitor cells; satellite cell; skeletal muscle.

FIG. 1.

Summary of experimental design. To compare constructs fabricated from different muscle sources, we cultured sheep muscle isolates and evaluated the development, structure, and function of our SMUs at different time points throughout the fabrication process. These assessments were completed on each of four experimental groups: zygomaticus major, masseter, soleus, and SM muscle sources. MDM, muscle differentiation medium; MGM, muscle growth medium; SM, semimembranosus; SMU, skeletal muscle unit.

FIG. 2.

Tissue digestion efficiency. (A) After allowing the tissue to enzymatically digest for 2.25 h, the undigested tissue was filtered out and weighed to provide insight into the digestion efficiency of each muscle group. There was a statistically significant difference in digestion efficiency between groups (p < 0.0001, n = 14 for ZM and n = 16 for others), and all groups were significantly different from each other. Symbols indicate statistically significant differences: □ from zygomaticus major, # from masseter, Δ from soleus, and ● from SM. (B) Upon completion of enzymatic digestion, cell counts were taken and normalized to the mass of muscle digested. There was a statistically significant difference in cell yield between groups (p = 0.0313, n = 10 for each group). The masseter yielded a significantly higher number of cells than the ZM (p = 0.0278). *p < 0.05. ZM, zygomaticus major.

FIG. 3.

Characterization of the isolated cell populations. The isolated cells were characterized to determine the percentage of (A) myogenic cells (expressing Pax7 and/or MyoD) and (B) mesenchymal cells (expressing vimentin), and (C) the number of cells expressing both Pax7/MyoD and vimentin present in the cell isolates. (A) There was no significant difference in the percentage of myogenic cells between groups (p = 0.7525, n = 2 for soleus and n = 4 for others). (B) There was no significant difference in the percentage of vimentin⁺ cells between groups (p = 0.5596, n = 2 for soleus, n = 3 for SM, and n = 4 for others). (C) There was no significant difference in the percentage of cells expressing both Pax7/MyoD and vimentin between groups (p = 0.4802, n = 2 for soleus and n = 4 for others).

FIG. 4.

Myogenic proliferation and early differentiation. (A) A BrdU assay coupled with immunostaining for MyoD was completed 4 days after initial plating. There were no significant differences in the number of MyoD⁺/mm² cells between groups, but there was a significant difference in BrdU⁺ cells between groups (p = 0.0093, n = 9 for soleus and n = 11 for others). The SM plates had a significantly higher number of BrdU⁺ cells/mm² than both the zygomaticus major (p = 0.0089) and the masseter (p = 0.0037) on day 4. (B) Immunostaining for myogenin completed 6 days after initial plating. There was a statistically significant difference in the number of myogenin⁺ cells/mm² between groups (p = 0.0292, n = 10 for ZM, n = 11 for masseter, n = 12 for soleus, and n = 13 for SM), with the zygomaticus major having a statistically higher number of myogenin⁺ cells/mm² than the masseter (p = 0.0326). *p < 0.05, **p < 0.01.

FIG. 5.

Late differentiation. Light microscopy images of the monolayers were taken before 3D construct formation to visualize the development of the myotubes. The images depicted show representative 10 × images of monolayers fabricated from (A) zygomaticus major, (B) masseter, (C) soleus, and (D) SM muscle sources. As can be noted from the images, the zygomaticus major group exhibited a less dense myotube network and more fibroblast overgrowth compared to the other groups. Scale bars = 500 μm. (E, F) Images such as these were used to evaluate myotube size (diameter) and density (number of myotubes/mm²) on day 11. (E) There was a significant difference in myotube size between groups (p = 0.0002, n = 6 for each group). The masseter had significantly larger myotubes than the ZM (p = 0.0002) and the SM (p = 0.0034). The soleus also had significantly larger myotubes than the ZM (p = 0.0202). (F) There was also a significant difference in the number of myotubes/mm² between groups (p = 0.0005, n = 6 for each group). The SM had significantly more myotubes/mm² than the ZM (p = 0.0029), soleus (p = 0.0055), and masseter (p = 0.0007). *p < 0.05, **p < 0.01, ***p < 0.001.

FIG. 6.

Maximum tetanic force production. Isometric force production in response to a tetanic electrical stimulus was measured 24 h after 3D construct formation. There was a significant difference in force production between groups (p = 0.0156, n = 22 for zygomaticus major, n = 26 for soleus, and n = 25 for others). The SMUs fabricated from masseter produced significantly more force than those fabricated from the zygomaticus major (p = 0.0110). *p < 0.05.

FIG. 7.

Visualization of myotubes within 3D SMUs. Histology was conducted on cross-sections of the three-dimensional constructs. The images depicted show representative images of SMUs fabricated from (A, E, I) zygomaticus major, (B, F, J) masseter, (C, G, K) soleus, and (D, H, L) SM muscle sources. Masson's trichrome (A–D) was performed to visualize collagen (blue), as well as muscle (red) and other cellular material (red). IHC (E–L) was performed to visualize the presence of laminin protein (green), muscle fibers (MF20, red), and cell nuclei (DAPI, blue). As can be noted from the images, the SMU fabricated from the zygomaticus major appeared to exhibit hypercellularity and less defined laminin staining compared to the other groups. Scale bars = 500 μm.