Impact of Human Epidermal Growth Factor on Tissue-Engineered Skeletal Muscle Structure and Function

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.

Introduction

Traumatic extremity injuries, surgical removal of skeletal muscle, and congenital defects can all result in a clinical condition known as volumetric muscle loss (VML).^1,2 VML is defined as skeletal muscle loss that exceeds the native regenerative capacity and results in permanent loss of muscle structure and function.³ VML frequently necessitates surgical intervention involving muscle flap or graft transposition.³ These techniques are limited by graft source availability, donor-site morbidity, and inability to appropriately regenerate muscle mass or effectively restore contractile function.⁴

Skeletal muscle tissue engineering technologies aim to grow exogenous muscle tissue that can replace lost native skeletal muscle while also promoting graft integration and muscle regeneration in vivo.^3,5 A significant obstacle hindering the translation of such technologies into medical therapies has been the limited success in developing human cell-sourced engineered skeletal muscle with in vitro structure or contractile forces comparable to native adult human muscle.^6–8

Skeletal muscle tissue engineering methodologies share the fundamental approach of creating in vitro environments simulating in vivo conditions, which enhance myogenesis by inducing primary skeletal muscle precursor cells down a myogenic lineage through the use of scaffold systems, mechanical forces, and/or chemical cues.⁸ Using these approaches, tissue engineers rely on different media and growth factor formulations during the in vitro culture of myogenic cells to control cell proliferation and differentiation.^9,10

The expansion of myogenic cells to sufficient numbers for tissue engineering requires the cell culture growth medium to contain serum and growth factors, such as fibroblast growth factor (FGF) that modulate cell proliferation and delay the onset of terminal differentiation.^10–12 For the differentiation of myogenic cells and for myotube fusion, the medium contains significantly less serum.^10–12 The absence of mitogenic components and inclusion of differentiation stimulating growth factors, such as insulin, trigger and promote differentiation.^10,11Myogenic cell proliferation and differentiation behavior is highly sensitive to the concentrations and timing of serum and growth factor administration.¹⁰

Furthermore, research suggests species-specific differences in myogenic cell response to certain growth factors and sera indicating that myogenic cells from different species have distinct growth factor requirements for optimal proliferation and differentiation.^9–10 The sensitivity of myogenic cells to these environmental factors can make it challenging to determine the optimal medium formulation and growth factor dosing strategy necessary for maximum cell performance in culture, especially when transitioning tissue engineering methodologies from animal to human cell culture models. Identifying essential growth factors, optimal concentration, and optimal exposure timing for human myogenic cell cultures is critical in enhancing the in vitro myogenesis of skeletal muscle precursors used in engineered skeletal muscle.

One growth factor of interest is human epidermal growth factor (hEGF), a mitogenic polypeptide found in platelets. hEGF is believed to be a key regulator of tissue growth and wound healing.^13–16 The signaling pathway for hEGF in native skeletal muscle has not been completely elucidated, but it is thought that hEGF regulates leukemia-inhibiting factor, a multifunctional cytokine involved in the regeneration of injured muscle.^17,18 Also, in human myogenic cell culture, hEGF has been shown to enhance proliferation by triggering cell division and metabolic processes.^19,20 The addition of hEGF to differentiation media has resulted in myotubes, which exhibit visible sarcomeric striations and spontaneous twitching.²⁰ Thus, in the development of techniques to engineered skeletal muscle, hEGF has been primarily used in human myogenic cell growth media.^6,19

Human myogenic cells expanded in medium treated with hEGF before being seeded in scaffolds have been shown to form myobundles, bundles of muscle fibers, with superior contractile function compared with cells expanded in untreated medium.⁶ To date, hEGF has been shown to have profound effects on human myogenic cell cultures in vitro, but the quantitative impact hEGF-treated growth and differentiation media has on engineered skeletal muscle structure and contractile function has yet to be evaluated.

Our laboratory has fabricated scaffold-free tissue-engineered skeletal muscle units (SMUs) from primary myogenic cells and fibroblasts.^1,12,21 Our SMU fabrication process is well defined in rat and ovine models, but this is the first publication detailing the fabrication of a human SMU comprising human primary cells. The scaffold-free design provides a unique opportunity to investigate the impact of hEGF on primary myogenic cell behavior and extracellular matrix deposition without the confounding effects caused by the chemical and mechanical cues present in different scaffold materials.

We hypothesized that exposing primary myogenic cells to hEGF in the SMU fabrication process through hEGF-treated muscle growth media and hEGF-treated muscle differentiation media would allow us to determine the optimal time points and time spans for enhancing in vitro myogenesis and skeletal muscle development. Thus, the purpose of this study was to examine the effects of hEGF on the structure and function of our scaffold-free SMU graft.

Materials and Methods

Isolation of muscle cells

Human soleus skeletal muscle surgical discards were obtained from two healthy males, 21 and 35 years of age. All experimental protocols involving the use of human skeletal muscle biopsies were approved by the University of Michigan Medical School Institutional Review Board (HUM00151617). Skeletal muscle biopsies underwent cell isolation procedures, as described previously.^1,12,21,22 In brief, muscle biopsies were removed under aseptic conditions, sterilized in 70% ethanol, and finely minced before being placed under ultraviolet light for 15 minutes. Minced muscle was then incubated in dissociation solution consisting of 2.3 mg/mL dispase (Cat. No. 17105-041; Thermo Fisher) and 0.3 mg/mL collagenase type IV (Cat. No. 17104-019; Thermo Fisher) for 2 hours.

The resulting suspension was then filtered through 100 μm (Cat. No. 22-363-549; Fisher Scientific) and 40 μm (Cat. No. 22-363-547; Fisher Scientific) mesh filters before undergoing centrifugation. The resulting cell pellet was either resuspended in muscle growth media (MGM) for immediate plating and SMU fabrication, or frozen down in freezing media for future use.

MGM contained 60% F-12 Kaighn's Medium (Cat. No. 21127-022; Gibco), 24% Dulbecco's modified Eagle's medium (DMEM; Cat. No. 11995-065; Gibco), 15% fetal bovine serum (FBS; Cat. No. 10437-028; Gibco), and 1% antibiotic/antimycotic (ABAM, Cat. No. 15240-062; Gibco). Additionally, MGM was supplemented with 2.4 ng/mL basic FGF (Cat. No. 100-18B; PeproTech) and 4.0 ng/mL dexamethasone (DEX; Cat. No. D8893; Sigma-Aldrich). Freezing media contained 20% horse serum (Cat. No. 16050122; Gibco), 10% dimethyl sulfoxide (Cat. No. BP231-100; Fisher Scientific), and 70% DMEM. The freezing medium was supplemented with 1% ABAM.

SMU fabrication and hEGF addition

Muscle cell isolates were plated in MGM on 60-mm tissue culture-treated polystyrene plates (BD Falcon, Franklin Lakes, NJ) at a seeding density of 10,000 cells/cm². To allow for cell attachment to the plates, cells were left undisturbed for 3 days before being fed MGM every 2 days. Seven days postseeding, after cells had become confluent and myotube networks had formed, the media was switched to muscle differentiation media (MDM). Cells were fed with MDM every 2 days for the duration of cell culture and SMU fabrication. MDM consisted of 70% Medium 199 (Cat. No. 1150067; Gibco), 23% DMEM, 6% FBS, and 1% ABAM. Additionally, MDM was supplemented with 0.1% insulin/transferrin/selenium X (Cat. No. 51500056; Gibco), 14.5 ug/mL ascorbic acid 2-phosphate (Cat. No. A8960-5G; Sigma Life-Aldrich), and 4.0 ng/mL DEX.

After 7 days in MDM, monolayers were manually delaminated with cell scrapers and transferred to 60-mm Sylgard-coated plates. Minuten pins were inserted through the monolayers into the Sylgard 3 cm apart and used as anchors. Over the next 7 days, the monolayer fused into cylindrical muscle constructs, held at length by the Minuten pins.

During this SMU fabrication process, summarized in Figure 1, the media was treated with 7.5 nM hEGF (Cat. No. CC-4107; Lonza) at three different time spans: 0 to 6 days postseeding (MGM Only), 7 to 21 days postseeding (MDM Only), and 0 to 21 days postseeding (MGM/MDM). These time spans were selected to analyze the impact of hEGF on human primary muscle cell cultures during cell proliferation and differentiation phases. The 0- to 7-day time span aligned with the time period the cell cultures were in MGM and the 7- to 21-day time span aligned with the time period the cell cultures were in MDM. Untreated plates served as controls.

FIG. 1.

Experimental timeline. To determine the effects of hEGF on the structure and function of SMUs, the media was treated with hEGF at three different time spans: 0 to 6 days to study effects on proliferation, 7 to 21 days to study effects on differentiation and structural maturation, and 0 to 21 days to investigate the combined effects of adding hEGF to both MGM and MDM. hEGF, human epidermal growth factor; SMUs, skeletal muscle units.

Late differentiation: myotube size and density analysis

Myotube size and density were evaluated to investigate the impact of hEGF on late differentiation. Fourteen days after initial plating of isolated cells, immediately before manual delamination of monolayers, monolayers in each experimental group (n = 15) were imaged using light microscopy. Three to five representative images were taken for each 60-mm plate. Using the ImageJ software package, images were evaluated to determine average myotube diameter and myotube network density. Using the multipoint tool on the ImageJ software package, the total number of myotubes in each representative image was quantified and normalized by total image area to determine myotube network density. Additionally, ImageJ's line tool was used to measure the diameter of each individual myotube in all representative images to determine average myotube diameter.

Seven to eight days after the SMUs formed three-dimensional (3D) cylinders, they underwent maximum tetanic isometric force measurements to evaluate contractile function, as described previously.^1,12,21,22 In brief, the anchor pin on one end of the SMU was attached to an optical force transducer and released from the Sylgard. Throughout testing, SMUs were kept in MDM cell culture at 37°C. To provide a relatively uniform electrical field throughout the entire length of the construct, platinum wire electrodes were submerged in the MDM and placed on either side of the SMU.

Before the onset of each stimulation, the average baseline force of each SMU was measured to determine passive tension. Tetanic isometric forces were elicited using a stimulus of a 600 ms duration square wave train of 0.1 ms pulses at 1000 mA at 60, 90, and 129 Hz. Tetanic isometric force data were recorded and analyzed using LabVIEW 2012 (National Instruments). Maximum tetanic isometric force measurements were normalized by MF20-positive cross-sectional area (CSA) to determine the SMU-specific force.

Structure: immunohistochemical analysis

Immunohistochemical staining (IHC) was conducted on SMU sections to qualitatively and quantitatively assess construct muscle structure. Immediately after force production measurements, SMUs were coated in Tissue Freezing Medium (Cat. No. 15-183-36; Fisher Scientific), frozen in liquid nitrogen-chilled isopentane, and stored at −80°C until sectioning. Ten micrometer-thick cross-sectional and longitudinal SMU cryosections were mounted on microscopy slides.

Before immunostaining, the slides were fixed in methanol chilled to −20°C. For immunostaining, slides were washed in 0.1% Triton X-100 (Cat. No. T8787; Sigma) in Dulbecco's phosphate-buffered saline (PBST). Slides were then blocked with 3% bovine serum albumin (Cat. No. A2153-10G; Sigma-Aldrich) in PBST for 30 min at room temperature. Following the blocking step, sections were covered in primary antibodies diluted in blocking medium and left to incubate overnight at 4°C.

To evaluate muscle content and overall construct CSA, SMU cross-sectional cryosections underwent IHC with primary antibodies for myosin heavy chain (MF20; mouse monoclonal antibody 1:200 dilution; Cat. No. MF 20-c; Developmental Studies Hybridoma Bank) and laminin (rabbit polyclonal antibody 1:200 dilution; Cat. No. 7463; Abcam). To evaluate sarcomeric structure, SMU longitudinal cryosections underwent IHC with a primary antibody for α-actinin (rabbit polyclonal antibody 1:300 dilution; Cat. No. ab18061; Abcam).

After incubation, samples were washed in PBST before incubation in 1:500 dilutions of Alexa Fluor anti-mouse or anti-rabbit (Life Technologies) for 3 h at room temperature. Following another set of washes in PBST, slides were preserved in ProLong Gold with 4′,6-diamidino-2-phenylindole (DAPI; Cat. No. P36935; Invitrogen). Slides were imaged using a ZEISS Apotome Microscope.

Using the ImageJ software package to analyze cross-sectional cryosections, the total CSA and MF20-positive CSA of each SMU was quantified. To measure total CSA, ImageJ's freehand selection tool was used to select the outer boundaries of the SMU. The total area was measured and converted from pixels to microns using ImageJ's “set scale” and “measure” features. To measure MF20-positive CSA, ImageJ's analyze particles tool was used to measure all areas of the section that display red MF20-positive fluorescence. Maximum tetanic isometric force measurements were normalized by MF20-positive CSA to determine the SMU-specific force.

Statistical analysis

For all graphs, boxes and bars indicate mean ± standard error of the mean. Using GraphPad Prism software, statistical differences between experimental groups were assessed through a one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. Differences were considered significant at a p < 0.05.

Results

Impact of hEGF on muscle cell differentiation and myotube fusion

Light microscopy images of monolayers 14 days postseeding were used to analyze muscle cell differentiation and myotube fusion. Images indicate monolayers fed hEGF have a denser myotube network, thicker myotube structures, and less fibroblast overgrowth compared with untreated control (Fig. 2A–D).

FIG. 2.

Impact of hEGF on muscle cell differentiation and myotube fusion. Light microscopy images of the monolayer were taken just before delamination on day 14 postseeding. Representative 10 × images are shown above for untreated control (A), and monolayers exposed to hEGF-treated MGM (B), hEGF-treated MDM (C), and hEGF-treated MGM+MDM (D). All monolayer images were used to evaluate myotube size (E) and myotube density (F). Scale bars = 500 μm. Boxes and bars indicate mean ± standard error of the mean. Different data point formats are used to differentiate different cell sources: • for frozen cells from the soleus of a 21-year-old male, ♦ for fresh cells from the soleus of a 35-year-old male, and ▪ for frozen cells from the same 35-year-old male. Δ symbol above bars indicates statistically significant differences from control. One-way ANOVA indicated that hEGF had a significant impact on both myotube size and density (p = 0.0001 and p = 0.0111, respectively). Post hoc analysis demonstrated significant difference in myotube diameter between monolayers exposed to hEGF-treated MGM and untreated monolayer controls (p < 0.0001). There was also a significant difference in myotube diameters between monolayers exposed to hEGF-treated MDM and control monolayers (p = 0.0065). Additionally, monolayers fed hEGF-treated MGM+MDM had a significantly greater myotube density when compared with control (p = 0.0151). Monolayers fed hEGF-treated MGM+MDM displayed a denser myotube network and less fibroblast overgrowth compared with untreated control. ANOVA, analysis of variance.

Measurements of myotube size and density revealed that timing of hEGF dosing had a significant impact on monolayer development. Monolayers fed hEGF-treated MGM had a significantly greater average myotube diameter than untreated monolayer controls with averages of 24.1 ± 0.5 μm and 20.8 ± 0.4 μm, respectively (p < 0.001; Fig. 2E). Additionally, monolayers fed hEGF-treated MDM had an average myotube diameter of 23.2 ± 0.6 μm, significantly different from control monolayers (p = 0.007). Analysis of myotube network density (myotube/mm²) also showed significant difference between groups (p = 0.01; Fig. 2F). Administration of hEGF in MGM+MDM led to a significant increase in myotube density when compared with control (p = 0.02) with means of 12.3 ± 0.3 myotubes/mm² versus 10.7 ± 0.4 myotubes/mm², respectively.

Effects of hEGF on SMU contractile force

After 3D construct formation, SMUs underwent tetanic force production measurements to assess contractile function (Fig. 3A). Average isometric tetanic forces were 2.6 ± 1.3 μN, 10.4 ± 1.2 μN, 54.2 ± 6.7 μN, and 90.4 ± 7.6 μN for control, hEGF-treated MGM only, hEGF-treated MDM only, and hEGF-treated MGM+MDM groups, respectively. A one-way ANOVA indicated that there was a significant difference in maximum tetanic force production between experimental groups (p < 0.0001, n = 15 for all experimental groups) signifying that treatment with hEGF and its dose timing increase SMU contractile force. A post hoc multiple comparisons test indicated no significant difference in force production between SMUs receiving hEGF-treated MGM and untreated control SMUs (p = 0.07).

FIG. 3.

Effects of hEGF maximum isometric tetanic force production (A) and specific force (B). Boxes and bars indicate mean ± standard error of the mean. Different data point formats are used to differentiate different cell sources: • for frozen cells from the soleus of a 21-year-old male, ♦ for fresh cells from the soleus of a 35-year-old male, and ▪ for frozen cells from the same 35-year-old male. Symbols above graphs indicate statistically significant differences: Δ from Control, # from MGM only, and □ from MDM only. Analysis of SMU maximum isometric force production in response to a tetanic electrical stimulus and SMU-specific force indicated a significant effect of hEGF timing (p < 0.001 for both). Compared with control, SMUs exposed to hEGF-treated MGM+MDM resulted in a 35-fold increase in force production and a 20-fold increase in specific force (p < 0.001 for both).

When compared with control, SMUs in hEGF-treated MDM and hEGF-treated MGM+MDM groups showed a significant 20-fold and 35-fold increase in average maximum tetanic force production respectively (p < 0.0001 for both). Additionally, average maximum tetanic force production between all hEGF-treated experimental groups were significantly different (p < 0.0001 for all). Overall, maximal force production was observed in SMUs in the hEGF-treated MGM+MDM group.

SMU maximum tetanic isometric force measurements were divided by MF20-positive CSA to assess SMU specific force (Fig. 3B). Average specific forces were 3.47 × 10⁻³ ± 3.47 × 10⁻³ N/cm², 6.47 × 10⁻³ ± 1.21 × 10⁻³ N/cm², 42.36 × 10⁻³ ± 9.45 × 10⁻³ N/cm², and 70.04 × 10⁻³ ± 8.58 × 10⁻³ N/cm² for control, hEGF-treated MGM only, hEGF-treated MDM only, and hEGF-treated MGM+MDM groups respectively. A one-way ANOVA indicated that there was a significant difference in specific force between experimental groups (p < 0.0001, n = 5 for all experimental groups). A post hoc multiple comparisons test indicated significant increases in specific force in SMUs receiving hEGF-treated MDM and SMUs receiving hEGF-treated MGM+MDM when compared with untreated control SMUs (p = 0.004 and p < 0.0001), respectively.

There was no significant difference in specific force between SMUs receiving hEGF-treated MGM and untreated control SMUs (p = 1.0). SMUs receiving hEGF-treated MGM had significantly lower specific forces compared with SMUs receiving hEGF-treated MDM (p = 0.007) and SMUs receiving hEGF-treated MGM+MDM (p < 0.0001). Additionally, there was a significant increase in specific force in hEGF-treated MGM+MDM SMUs compared with hEGF-treated MDM SMUs (p = 0.04). SMUs in hEGF-treated MGM+MDM groups showed the largest average specific force, 20 times larger than the average specific force of the control group.

Role of hEGF in SMU structural maturation

IHC analysis of SMU cross-sections were used to determine SMU composition and CSA. In all experimental groups, DAPI, MF20, and laminin costains indicated the presence of large multinucleated myotubes surrounded by laminin sheaths (Fig. 4A–D). Furthermore, IHC analysis indicated cell viability throughout the entire SMU cross-section in all groups, suggesting there were no limitations in nutrient diffusion during the SMU fabrication process.

FIG. 4.

Role of hEGF in SMU structural maturation. IHC of 3D SMU cross-sections was conducted to visualize cell viability (DAPI, blue), myosin heavy chain (MF20, red), and laminin protein (green). The images depicted are representative images of SMUs fed untreated media (A), hEGF-treated MGM (B), hEGF-treated MDM (C), and hEGF-treated MGM+MDM (D). The total CSA (E) and MF20-positive CSA (F) were quantified for each SMU cross-section that underwent staining (n = 5 per experimental group). Scale bars = 500 μm. Boxes and bars indicate mean ± standard error of the mean. Symbols above graphs indicate statistically significant differences: Δ from Control, and # from MGM only. Nucleated MF20-positive myotubes surrounded by laminin were present in all experimental groups. A one-way ANOVA indicated there was significant difference in CSA between groups (p = 0.0003). SMUs exposed to hEGF-treated MDM had significantly greater CSAs when compared with controls (p = 0.0028) and SMUs exposed to hEGF-treated MGM (p = 0.0004). SMUs fed hEGF-treated MGM+MDM had significantly greater CSAs compared with SMUs exposed to SMUs fed hEGF-treated MGM (p = 0.0230). There was also significant difference in myosin heavy chain content between experimental groups (p = 0.0022). SMUs exposed to hEGF showed significantly greater myosin heavy chain content compared with untreated controls (p = 0.0378 for hEGF-treated MGM, p = 0.0027 for hEGF-treated MDM, p = 0.0060 for hEGF-treated MGM+MDM). CSA, cross-sectional area; DAPI, 4′,6-diamidino-2-phenylindole; IHC, immunohistochemical staining.

A one-way ANOVA indicated that there was significant difference in CSA between experimental groups (p = 0.0003; Fig. 4E). SMUs exposed to hEGF-treated MDM had significantly greater whole construct CSAs compared with untreated control SMUs (p = 0.0028) with mean CSAs of 3.2 ± 0.1 mm² versus 2.3 ± 0.1 mm², respectively. Additionally, SMUs exposed to hEGF-treated MGM had mean CSAs of 2.1 ± 0.1 mm², significantly smaller than the mean CSAs of SMUs exposed to hEGF-treated MDM (p = 0.0004) and hEGF-treated MGM+MDM (2.8 ± 0.2 mm²; p = 0.02).

The MF20-positive CSA was also measured to quantify the amount of myosin heavy chain content in each SMU. A one-way ANOVA revealed that there was a statistically significant difference in MF20-positive CSA between groups (p = 0.002; Fig. 4F). A post hoc multiple comparisons test indicated all experimental groups exposed to hEGF showed a significant increase in myosin heavy chain content compared with control (p = 0.04 for hEGF-treated MGM, p = 0.003 for hEGF-treated MDM, p = 0.006 for hEGF-treated MGM+MDM).

SMUs receiving hEGF-treated MGM, hEGF-treated MDM, and hEGF-treated MGM+MDM had mean MF20-positive CSAs of 0.126 ± 0.02 mm², 0.151 ± 0.01 mm², and 0.144 ± 0.01 mm², respectively. Mean MF20-positive CSAs for hEGF-treated MGM and MGM+MDM SMUs are over two times greater than the untreated control mean CSA of 0.070 ± 0.01 mm².

Staining of longitudinal sections of 3D constructs with DAPI and α-actinin revealed myofilament alignment, the development of sarcomeric structure, and the maturing of Z-lines in hEGF-treated SMUs, a sarcomeric organization similar to adult skeletal muscle in situ (Fig. 5). While myofilament alignment was present in untreated control SMUs, the development of organized sarcomeric structure was not observed. The improved structural maturation of 3D SMUs treated with hEGF is likely correlated with the SMU's enhanced force production compared with untreated controls.

FIG. 5.

Impact of hEGF on sarcomeric structure. IHC of 3D SMU longitudinal sections was conducted to visualize sarcomeric structure (α-actinin, green). The images depicted are representative images of SMUs fed untreated media (A), hEGF-treated MGM (B), hEGF-treated MDM (C), and hEGF-treated MGM+MDM (D). Examples of sarcomeric striations are indicated by white arrows. Scale bars = 20 μm. Staining revealed the presence of organized sarcomeric structure and myofilament alignment in hEGF-treated SMUs, whereas organized sarcomeric structure was mostly absent from untreated controls. 3D, three-dimensional.

Discussion

Overall, this study used scaffold-free SMUs to investigate the impact of hEGF on the in vitro myogenesis and extracellular matrix deposition of human skeletal muscle stem cells in skeletal muscle tissue engineering models. We hypothesized that exposing myogenic cell cultures to 7.5 nM hEGF at different time spans during the fabrication process would determine the most favorable dosing strategy for enhancing human SMU structure and function.

The hEGF time periods examined aligned with periods of myogenic cell proliferation and differentiation in culture. Cell cultures exposed to hEGF-treated MGM were introduced to the growth factor during the key myogenic cell proliferation phase, 0 to 6 days postseeding. Monolayers fed with hEGF-treated MDM were stimulated by the growth factor during periods of myogenic cell differentiation and maturation in both monolayer (two-dimensional [2D]) and SMU (3D) form, 7 to 21 days postseeding. The addition of hEGF during the myogenic proliferation phase (MGM only) or during myocyte differentiation (MDM only) led to myotube hypertrophy and structural maturation during late differentiation in monolayers, indicated by the increased myotube sizes compared with control.

Interestingly, hEGF-treated MGM+MDM cultures did not display an increase in myotube size, whereas hEGF-treated MGM only and hEGF-treated MDM only cultures did not show a significant increase in myotube density. Previous literature indicates that hEGF promotes myoblast proliferation but inhibits myoblast differentiation, providing a potential explanation for such results.^23,24 The sustained administration of hEGF in hEGF-treated MGM+MDM cultures could have inhibited the fusion of myofibers to each other, diminishing overall myotube size. Periods of no hEGF administration in hEGF-treated MGM and hEGF-treated MDM experimental groups would have allowed for the fusion of myofibers to each other, increasing myotube size but decreasing myotube density.

Further analysis is necessary to determine which steps in the differentiation process are inhibited by hEGF. In total, these myotube diameter and myotube density results indicate that hEGF enhances human muscle cell differentiation and myotube fusion in 2D monolayers.

The addition of hEGF to cell culture also greatly impacted 3D SMU structure. All experimental groups treated with hEGF resulted in SMUs with advanced structural maturation compared with control, as indicated by the presence of organized sarcomeric structures. Additionally, the multinucleated myofibers surrounded by laminin sheaths observed in SMU cross-sections are physiologically similar to the individual muscle fibers surrounded by basal lamina sheaths in native muscle tissue.

The increase in myosin content indicates that the SMUs treated with hEGF had significantly increased myofibril content compared with control. hEGF additionally led to hEGF-treated MDM only SMUs and hEGF-treated MGM+MDM SMUs having significantly larger CSAs compared with hEGF-treated MGM only SMUs, suggesting an increase in extracellular matrix proteins and other cellular material in addition to the increase in myosin content. The significant increase in myosin heavy chain and organized sarcomeric structure likely contributed to the increased force production and specific force of SMUs in hEGF-treated MDM and hEGF-treated MGM+MDM groups.

Our data suggest that starting time point and duration of treatment of hEGF play a significant role in SMU contractile function. The structural development induced by hEGF in 2D monolayers and 3D SMUs contributed to greater SMU force production, most notably in hEGF-treated MGM+MDM SMUs. While hEGF may have inhibited overall myotube size in monolayers, the extracellular matrix deposition and organization induced by hEGF supported the muscle networks present in SMUs allowing for efficient and unified force transmission. Overall, sustained administration of 7.5 nM hEGF during myogenic cell proliferation, differentiation, and maturation phases resulted in SMUs capable of producing the greatest specific forces. Initial studies examining 1.5, 7.5, and 10 nM hEGF in MGM+MDM suggest that hEGF continues to enhance SMU contractile function within that range of concentrations.

Using our scaffold-free tissue engineering model, we demonstrated that the supplementation of hEGF to human primary myogenic cell cultures improved tissue-engineered skeletal muscle structural and functional characteristics with the most promising advancements occurring in cultures exposed to hEGF during the entire fabrication process. Our results suggest that hEGF serves as a critical growth factor in advancing human skeletal muscle-engineered tissue models and developing skeletal muscle with adult phenotypes.

Disclosure Statement

No competing financial interests exist.

Funding Information

The authors would like to acknowledge the support of the Department of Defense (W81XWH-16-1-0752) and the National Institute of Dental and Craniofacial Research (T32-DE007057-39).