Engineering Skeletal Muscle Grafts with PAX7::GFP-Sorted Human Pluripotent Stem Cell-Derived Myogenic Progenitors on Fibrin Microfiber Bundles for Tissue Regeneration.

Tissue engineering strategies that combine human pluripotent stem cell-derived myogenic progenitors (hPDMs) with advanced biomaterials provide promising tools for engineering 3D skeletal muscle grafts to model tissue development in vitro and promote muscle regeneration in vivo. We recently demonstrated (i) the potential for obtaining large numbers of hPDMs using a combination of two small molecules without the overexpression of transgenes and (ii) the application of electrospun fibrin microfiber bundles for functional skeletal muscle restoration following volumetric muscle loss. In this study, we aimed to demonstrate that the biophysical cues provided by the fibrin microfiber bundles induce hPDMs to form engineered human skeletal muscle grafts containing multinucleated myotubes that express desmin and myosin heavy chains and that these grafts could promote regeneration following skeletal muscle injuries. We tested a genetic PAX7 reporter line (PAX7::GFP) to sort for more homogenous populations of hPDMs. RNA sequencing and gene set enrichment analyses confirmed that PAX7::GFP-sorted hPDMs exhibited high expression of myogenic genes. We tested engineered human skeletal muscle grafts derived from PAX7::GFP-sorted hPDMs within in vivo skeletal muscle defects by assessing myogenesis, engraftment and immunogenicity using immunohistochemical staining. The PAX7::GFP-sorted groups had moderately high vascular infiltration and more implanted cell association with embryonic myosin heavy chain (eMHC) regions, suggesting they induced pro-regenerative microenvironments. These findings demonstrated the promise for the use of PAX7::GFP-sorted hPDMs on fibrin microfiber bundles and provided some insights for improving the cell-biomaterial system to stimulate more robust in vivo skeletal muscle regeneration.

Protocol for the Use of a Novel Bioreactor System for Hydrated Mechanical Testing, Strained Sterile Culture, and Force of Contraction Measurement of Tissue Engineered Muscle Constructs.

Bioreactor systems are built as controlled environments for biological processes and utilized in the field of tissue engineering to apply mechanical, spatial, and chemical cues to developing tissue grafts. Often the systems are applied to instruct differentiation and maturation of the cells grown inside. Perhaps the most obvious targets for strain and compression-based bioreactors are mechanically active tissues, as it is hypothesized that biomimetic mechanical environments instruct immature cells to form differentiated tissues. One such tissue, skeletal muscle, has been identified as a key candidate for strain application due to the close structure-function relationship of myofibers. Here we detail the multiple uses of a custom-built bioreactor system in combination with electrospun fibrin microfibers for muscle tissue engineering. Outlined below are the methods used in the system to test the mechanical properties of hydrogel-based scaffolds in an aqueous environment, including Young's modulus and poroelasticity. Additionally, we demonstrate the application of tensile strain to sterile cell cultures grown on electrospun scaffolds and perform end-point testing of tissue contractility with the addition of an electrode.

Modeling the mechanics of fibrous-porous scaffolds for skeletal muscle regeneration.

The scaffolds for skeletal muscle regeneration are designed to mimic the structure, stiffness, and strains applied to the muscle under physiologic conditions. The external strains are also used to stimulate myogenesis of the (stem) cells seeded on the scaffold. The time- and location-dependent mechanics inside the scaffold determine the microenvironment for the seeded cells. Here, fibrous-porous cylindrical scaffolds under the action of external cyclic strains are considered. The scaffold mechanics are described as two-phase (poroelastic) where the solid phase is associated with the fibers and the fluid phase is associated with the liquid-containing pores. In response to an applied cyclic strain, pressure oscillates and fluid moves radially toward and away from the axis of the scaffold. We compute the directions and magnitudes of the radial gradients of the poroelastic characteristics (solid-phase displacement, strain, and velocity; fluid-phase pressure and velocity; relative fluid-solid-phase velocity) determined by the boundary conditions and geometry of the scaffold. Several kinds of the external cyclic strain are analyzed and the resulting poroelastic functions are found. The poroelastic characteristics are obtained in closed form which is convenient for further consideration of myogenesis of the seeded cells and ultimately for the design of the scaffolds for skeletal muscle regeneration. Graphical abstract.

Engineering 3D skeletal muscle primed for neuromuscular regeneration following volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms the native regenerative capabilities of skeletal muscle and has few effective treatments to regain lost muscle mass and function. Tissue engineered muscle constructs designed to promote neuromuscular regeneration are a promising therapeutic avenue. To date, there has been no engineered muscle construct for VML treatment that has incorporated a pharmacologic agent to promote neuromuscular regeneration. Here, we have modified electrospun fibrin microfiber bundles, which have demonstrated muscle regenerative potential, with the heparan sulfate proteoglycan, agrin, to stimulate innervation post-VML. Myoblasts cultured on microfiber bundles with either soluble or chemically tethered agrin demonstrated statistically significant increased clustering of acetylcholine receptors (AChRs) with soluble agrin displaying AChR clusters throughout the myofiber bundles, and tethered agrin displaying AChR clusters only at 10 µm from the substrate surface. Following implantation into murine VML defects for 4 weeks, constructs pre-treated with soluble or tethered agrin resulted in statistically significant increased neuromuscular junctions, regenerating myofibers, vascular infiltration, neural infiltration, and nuclear yes-associated protein (YAP) expression within the defect site compared to the control without agrin. The agrin-tethered microfiber bundles provided sustained agrin signaling within the regenerating site during the 4-week post-implantation periods and further augmented the density of regenerating myofibers in regenerated tissue with statistical significance compared to constructs with soluble agrin. These data demonstrate the neuromuscular regenerative potential of engineered muscle constructs pre-treated to induce AChR clustering with locally delivered agrin at the site of VML regeneration.

Modified cell-electrospinning for 3D myogenesis of C2C12s in aligned fibrin microfiber bundles.

Electrospinning methods can generate scaffolds with alignment cues to guide the development of myogenic precursors into 3D skeletal muscle grafts. However, cells seeded onto these scaffolds adhere to the exterior resulting in regions of acellularity within the scaffold interior. To overcome this limitation, we modified an aqueous solution-electrospinning method to encapsulate C2C12s and electrospin them into fibrin/polyethylene oxide (PEO) microfiber bundles. We demonstrated that loading C2C12s as cellular aggregates (80-90 µm in diameter) and modifying several other electrospinning parameters dramatically increased cell viability following exposure to the 4.5 kV electric field. C2C12-seeded fibrin/PEO microfiber bundles were cultured for up to seven days. Uninduced and myogenically induced C2C12s proliferated, elongated and became multinucleated. Myogenic induction increased the number of myotube-associated nuclei (36.4 ±â€¯12% vs. 6.2 ±â€¯1.9%), myotube length (122.4 ±â€¯10.9 µm vs. 59.9 ±â€¯8.3 µm), and myotube diameter (16.76 ±â€¯2.06 µm vs. 12.49 ±â€¯0.93 µm). The data presented in this study demonstrates for the first time that cells can be loaded inside the aligned fibrin hydrogel 3D construct during aqueous solution electrospinning while retaining their potential for de novo tissue formation.

Myoblast maturity on aligned microfiber bundles at the onset of strain application impacts myogenic outcomes.

Engineered skeletal muscle grafts may be employed in various applications including the treatment of volumetric muscle loss (VML) and pharmacological drug screening. To recapitulate the well-defined structure of native muscle, tensile strains have been applied to the grafts. In this study, we cultured C2C12 murine myoblasts on electrospun fibrin microfiber bundles for 7 days in custom-built bioreactor units and investigated the impact of strain regimen and delayed onset of tensile straining on myogenic outcomes. The substrate topography induced uniaxial alignment of cells in all (strained and unstrained) groups. The engineered grafts in strained groups were subjected to 10% strain amplitude for 6 h per day. We found that both static and cyclic uniaxial strains resulted in similar morphological and gene expression outcomes. However, relative to 0% strain groups, there were stark increases in myotube diameter, myosin heavy chain (MHC) coverage, and expression of key myogenic genes (Pax 7, Troponin, MHC I, MHC IIb, MHC IIx) only if strain was applied at Days 5-7 rather than Days 3-7. This finding suggests that a critical indicator of myogenic improvement under strain in our system is the phenotype of the cells at the onset of strain and suggests that this is a key parameter that should be considered in studies where myoblasts are subjected to biophysical stimulation to promote tissue formation. STATEMENT OF SIGNIFICANCE: This is the first report on the impact of the timing of the initial application of mechanical strain for improving the myogenic outcomes of 3D engineered skeletal muscle grafts. In this work, immature skeletal myoblasts were grown on topographically aligned, electrospun fibrin microfiber bundles and we applied 10% uniaxial static or cyclic strain. We concluded that the maturity of myoblasts prior to strain application, rather than strain waveform, was the primary predictor of improved myogenic outcomes, including myogenic gene expression and myotube morphology. Elucidating the optimal conditions for strain application is a vital step in recapitulating physiological myogenic properties in tissue engineered skeletal muscle constructs, with applications for treating volumetric muscle loss, disease modeling, and drug testing.

Engineering functional and histological regeneration of vascularized skeletal muscle.

Tissue engineering strategies to treat patients with volumetric muscle loss (VML) aim to recover the structure and contractile function of lost muscle tissue. Here, we assessed the capacity of novel electrospun fibrin hydrogel scaffolds seeded with murine myoblasts to regenerate the structure and function of damaged muscle within VML defects to the mouse tibialis anterior muscle. The electrospun fibrin scaffolds provide pro-myogenic alignment and stiffness cues, myomimetic hierarchical structure, suturability, and scale-up capabilities. Myoblast-seeded scaffolds enabled remarkable muscle regeneration with high myofiber and vascular densities after 2 and 4 weeks, mimicking that of native skeletal muscle, while acellular scaffolds lacked muscle regeneration. Both myoblast-seeded and acellular scaffolds fully recovered muscle contractile function to uninjured values after 2 and 4 weeks. Electrospun scaffolds pre-vascularized with co-cultured human endothelial cells and human adipose-derived stem cells implanted into VML defects for 2 weeks anastomosed with host vasculature and were perfused with host red blood cells. These data demonstrate the significant potential of electrospun fibrin scaffolds seeded with myoblasts to fully regenerate the structure and function of volumetric muscle defects and these scaffolds offer a promising treatment option for patients with VML.

A Poroelastic Model of a Fibrous-Porous Tissue Engineering Scaffold.

Tissue engineering scaffolds are used in conjunction with stem cells for the treatment of various diseases. A number of factors provided by the scaffolds affect the differentiation of stem cells. Mechanical cues that are part of the natural cellular microenvironment can both accelerate the differentiation toward particular cell lineages or induce differentiation to an alternative cell fate. Among such factors, there are externally applied strains and mechanical (stiffness and relaxation time) properties of the extracellular matrix. Here, the mechanics of a fibrous-porous scaffold is studied by applying a coordinated modeling and experimental approach. A force relaxation experiment is used, and a poroelastic model associates the relaxation process with the fluid diffusion through the fibrous matrix. The model parameters, including the stiffness moduli in the directions along and across the fibers as well as fluid diffusion time, are estimated by fitting the experimental data. The time course of the applied force is then predicted for different rates of loading and scaffold porosities. The proposed approach can help in a reduction of the technological and experimental efforts to produce 3-D scaffolds for regenerative medicine as well as in a higher accuracy of the estimation of the local factors sensed by stem cells.

Biophysical Stimulation for Engineering Functional Skeletal Muscle.

Tissue engineering is a promising therapeutic strategy to regenerate skeletal muscle. However, ex vivo cultivation methods typically result in a low differentiation efficiency of stem cells as well as grafts that resemble the native tissues morphologically, but lack contractile function. The application of biomimetic tensile strain provides a potent stimulus for enhancing myogenic differentiation and engineering functional skeletal muscle grafts. We reviewed integrin-dependent mechanisms that potentially link mechanotransduction pathways to the upregulation of myogenic genes. Yet, gaps in our understanding make it challenging to use these pathways to theoretically determine optimal ex vivo strain regimens. A multitude of strain protocols have been applied to in vitro cultures for the cultivation of myogenic progenitors (adipose- and bone marrow-derived stem cells and satellite cells) and transformed murine myoblasts, C2C12s. Strain regimens are characterized by orientation, amplitude, and time-dependent factors (effective frequency, duration, and the rest period between successive strain cycles). Analysis of published data has identified possible minimum/maximum values for these parameters and suggests that uniaxial strains may be more potent than biaxial strains, possibly because they more closely mimic physiologic strain profiles. The application of these biophysical stimuli for engineering 3D skeletal muscle grafts is nontrivial and typically requires custom-designed bioreactors used in combination with biomaterial scaffolds. Consideration of the physical properties of these scaffolds is critical for effective transmission of the applied strains to encapsulated cells. Taken together, these studies demonstrate that biomimetic tensile strain generally results in improved myogenic outcomes in myogenic progenitors and differentiated myoblasts. However, for 3D systems, the optimization of the strain regimen may require the entire system including cells, biomaterials, and bioreactor, to be considered in tandem.

Characterization of a novel bioreactor system for 3D cellular mechanobiology studies.

In vitro engineering systems can be powerful tools for studying tissue development in response to biophysical stimuli as well as for evaluating the functionality of engineered tissue grafts. It has been challenging, however, to develop systems that adequately integrate the application of biomimetic mechanical strain to engineered tissue with the ability to assess functional outcomes in real time. The aim of this study was to design a bioreactor system capable of real-time conditioning (dynamic, uniaxial strain, and electrical stimulation) of centimeter-long 3D tissue engineered constructs simultaneously with the capacity to monitor local strains. The system addresses key limitations of uniform sample loading and real-time imaging capabilities. Our system features an electrospun fibrin scaffold, which exhibits physiologically relevant stiffness and uniaxial alignment that facilitates cell adhesion, alignment, and proliferation. We have demonstrated the capacity for directly incorporating human adipose-derived stromal/stem cells into the fibers during the electrospinning process and subsequent culture of the cell-seeded constructs in the bioreactor. The bioreactor facilitates accurate pre-straining of the 3D constructs as well as the application of dynamic and static uniaxial strains while monitoring bulk construct tensions. The incorporation of fluorescent nanoparticles throughout the scaffolds enables in situ monitoring of local strain fields using fluorescent digital image correlation techniques, since the bioreactor is imaging compatible, and allows the assessment of local sample stiffness and stresses when coupled with force sensor measurements. In addition, the system is capable of measuring the electromechanical coupling of skeletal muscle explants by applying an electrical stimulus and simultaneously measuring the force of contraction. The packaging of these technologies, biomaterials, and analytical methods into a single bioreactor system has produced a powerful tool that will enable improved engineering of functional 3D ligaments, tendons, and skeletal muscles. Biotechnol. Bioeng. 2016;113: 1825-1837. © 2016 Wiley Periodicals, Inc.