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.

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.

Vascularized and Innervated Skeletal Muscle Tissue Engineering.

Volumetric muscle loss (VML) is a devastating loss of muscle tissue that overwhelms the native regenerative properties of skeletal muscle and results in lifelong functional deficits. There are currently no treatments for VML that fully recover the lost muscle tissue and function. Tissue engineering presents a promising solution for VML treatment and significant research has been performed using tissue engineered muscle constructs in preclinical models of VML with a broad range of defect locations and sizes, tissue engineered construct characteristics, and outcome measures. Due to the complex vascular and neural anatomy within skeletal muscle, regeneration of functional vasculature and nerves is vital for muscle recovery following VML injuries. This review aims to summarize the current state of the field of skeletal muscle tissue engineering using 3D constructs for VML treatment with a focus on studies that have promoted vascular and neural regeneration within the muscle tissue post-VML.

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.

Adipose-derived Stem/Stromal Cells on Electrospun Fibrin Microfiber Bundles Enable Moderate Muscle Reconstruction in a Volumetric Muscle Loss Model.

Current treatment options for volumetric muscle loss (VML) are limited due to donor site morbidity, lack of donor tissue, and insufficient functional recovery. Tissue-engineered skeletal muscle grafts offer the potential to significantly improve functional outcomes. In this study, we assessed the potential pro-myogenic effects of human adipose-derived stem cells (ASCs) seeded onto electrospun uniaxially aligned fibrin hydrogel microfiber bundles. Although both uninduced and 5-azacytidine-induced ASCs exhibited alignment, elongation, and diffuse muscle marker expression when grown on microfiber bundles for 2 months in vitro, both groups failed to fully recapitulate myotube characteristics. To assess the muscle regeneration potential of ASCs in vivo, ASC-seeded fibrin microfiber bundles were implanted in a robust murine VML defect model. Minimal fibrosis was observed surrounding implanted acellular hydrogel fibers at 2 and 4 weeks, and fibers seeded with ASCs exhibited up to 4 times higher volume retention than acellular fibers. We observed increased numbers of cells positive for the regenerating muscle marker embryonic myosin and the mature muscle marker myosin heavy chain in ASC-seeded fibers compared with acellular fibers at 1 and 3 months post-transplantation. Regenerating muscle cells were closely associated with ASC-derived cells and in some cases had potentially fused with them. These findings demonstrate that despite failing to undergo myogenesis in vitro, ASCs combined with electrospun fibrin microfibers moderately increased muscle reconstruction in vivo compared with acellular fibers following a severe VML defect.

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.

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.