Abstract
Skeletal muscle tissue engineering technologies have the potential to treat volumetric muscle loss (VML) by growing exogenous muscle tissue. However, there has been limited success in engineering human cell-sourced skeletal muscle with structure and function comparable to native adult human muscle. The use of growth factors at optimal concentrations and delivery times is critical in enhancing the in vitro myogenesis of satellite cells used in engineered skeletal muscle. The mitogenic protein human epidermal growth factor (hEGF) is of particular interest because it enhances satellite cell proliferation and sarcomeric structure formation in myogenic cell cultures. In this study, we used our scaffold-free tissue-engineered skeletal muscle units (SMUs) to examine the effects of hEGF on the structure and function of human cell-sourced engineered skeletal muscle. During our established SMU fabrication process, human muscle cell isolates were exposed to media treated with 7.5 nM hEGF at three different time spans during the 21-day cell culture period: 0 to 6 days postseeding (hEGF-treated Muscle Growth Media [MGM] Only), 7 to 21 days postseeding (hEGF-treated Muscle Differentiation Media (MDM) Only), and 0 to 21 days postseeding (hEGF-treated MGM+MDM). Control cell cultures were fed standard MGM and MDM (no hEGF treatment). During the fabrication process, light microscopy was used to examine proliferation and differentiation of myogenic cells in the monolayer. After SMU formation, the three-dimensional constructs underwent tetanic force production measurements to evaluate contractile function and immunohistochemical staining to examine SMU structure. Results indicated that hEGF administration impacted myogenesis, by increasing myotube diameter in hEGF-treated MGM only and hEGF-treated MDM-only cell cultures, and by increasing myotube density in hEGF-treated MGM+MDM cultures. The exposure of myogenic cells to hEGF during any time period of the fabrication process led to a significant increase in SMU myosin heavy-chain content. SMUs exposed to hEGF-treated MDM and hEGF-treated MGM+MDM exhibited greater cross-sectional areas and more organized sarcomeric structure. Furthermore, hEGF-treated MGM+MDM SMUs displayed significantly enhanced contractile function compared with controls, indicating advanced functional maturation. In conclusion, hEGF supplementation in human primary myogenic cell cultures advances tissue-engineered skeletal muscle structural and functional characteristics. Impact statement Our research suggests that human epidermal growth factor (hEGF) serves as a critical growth factor in enhancing in vitro skeletal muscle cell proliferation and differentiation during myogenesis and advances human skeletal muscle engineered tissues toward a more native adult skeletal muscle phenotype. Understanding the impact of hEGF on engineered skeletal muscle function and structure is valuable in determining the optimal culture conditions for the development of tissue engineering-based therapies for volumetric muscle loss.