Skeletal muscle regeneration with robotic actuation-mediated clearance of neutrophils.

Mechanical stimulation (mechanotherapy) can promote skeletal muscle repair, but a lack of reproducible protocols and mechanistic understanding of the relation between mechanical cues and tissue regeneration limit progress in this field. To address these gaps, we developed a robotic device equipped with real-time force control and compatible with ultrasound imaging for tissue strain analysis. We investigated the hypothesis that specific mechanical loading improves tissue repair by modulating inflammatory responses that regulate skeletal muscle regeneration. We report that cyclic compressive loading within a specific range of forces substantially improves functional recovery of severely injured muscle in mice. This improvement is attributable in part to rapid clearance of neutrophil populations and neutrophil-mediated factors, which otherwise may impede myogenesis. Insights from this work will help advance therapeutic strategies for tissue regeneration broadly.

A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment.

In vitro systems that mimic organ functionality have become increasingly important tools in drug development studies. Systems that measure the functional properties of skeletal muscle are beneficial to compound screening studies and also for integration into multiorgan devices. To date, no studies have investigated human skeletal muscle responses to drug treatments at the single myotube level in vitro. This report details a microscale cantilever chip-based assay system for culturing individual human myotubes. The cantilevers, along with a laser and photo-detector system, enable measurement of myotube contractions in response to broad-field electrical stimulation. This system was used to obtain baseline functional parameters for untreated human myotubes, including peak contractile force and time-to-fatigue data. The cultured myotubes were then treated with known myotoxic compounds and the resulting functional changes were compared to baseline measurements as well as known physiological responses in vivo. The collected data demonstrate the system's capacity for screening direct effects of compound action on individual human skeletal myotubes in a reliable, reproducible, and noninvasive manner. Furthermore, it has the potential to be utilized for high-content screening, disease modeling, and exercise studies of human skeletal muscle performance utilizing iPSCs derived from specific patient populations such as the muscular dystrophies.

Timed Delivery of Therapy Enhances Functional Muscle Regeneration.

Cell transplantation is a promising therapeutic strategy for the treatment of traumatic muscle injury in humans. Previous investigations have typically focused on the identification of potent cell and growth factor treatments and optimization of spatial control over delivery. However, the optimal time point for cell transplantation remains unclear. Here, this study reports how myoblast and morphogen delivery timed to coincide with specific phases of the inflammatory response affects donor cell engraftment and the functional repair of severely injured muscle. Delivery of a biomaterial-based therapy timed with the peak of injury-induced inflammation leads to potent early and long-term regenerative benefits. Diminished inflammation and fibrosis, enhanced angiogenesis, and increased cell engraftment are seen during the acute stage following optimally timed treatment. Over the long term, treatment during peak inflammation leads to enhanced functional regeneration, as indicated by reduced chronic inflammation and fibrosis along with increased tissue perfusion and muscle contractile force. Treatments initiated immediately after injury or after inflammation had largely resolved provided more limited benefits. These results demonstrate the importance of appropriately timing the delivery of biologic therapy in the context of muscle regeneration. Biomaterial-based timed delivery can likely be applied to other tissues and is of potential wide utility in regenerative medicine.

Morphogenesis of 3D vascular networks is regulated by tensile forces.

Understanding the forces controlling vascular network properties and morphology can enhance in vitro tissue vascularization and graft integration prospects. This work assessed the effect of uniaxial cell-induced and externally applied tensile forces on the morphology of vascular networks formed within fibroblast and endothelial cell-embedded 3D polymeric constructs. Force intensity correlated with network quality, as verified by inhibition of force and of angiogenesis-related regulators. Tensile forces during vessel formation resulted in parallel vessel orientation under static stretching and diagonal orientation under cyclic stretching, supported by angiogenic factors secreted in response to each stretch protocol. Implantation of scaffolds bearing network orientations matching those of host abdominal muscle tissue improved graft integration and the mechanical properties of the implantation site, a critical factor in repair of defects in this area. This study demonstrates the regulatory role of forces in angiogenesis and their capacities in vessel structure manipulation, which can be exploited to improve scaffolds for tissue repair.

Biologic-free mechanically induced muscle regeneration.

Severe skeletal muscle injuries are common and can lead to extensive fibrosis, scarring, and loss of function. Clinically, no therapeutic intervention exists that allows for a full functional restoration. As a result, both drug and cellular therapies are being widely investigated for treatment of muscle injury. Because muscle is known to respond to mechanical loading, we investigated instead whether a material system capable of massage-like compressions could promote regeneration. Magnetic actuation of biphasic ferrogel scaffolds implanted at the site of muscle injury resulted in uniform cyclic compressions that led to reduced fibrous capsule formation around the implant, as well as reduced fibrosis and inflammation in the injured muscle. In contrast, no significant effect of ferrogel actuation on muscle vascularization or perfusion was found. Strikingly, ferrogel-driven mechanical compressions led to enhanced muscle regeneration and a ∼threefold increase in maximum contractile force of the treated muscle at 2 wk compared with no-treatment controls. Although this study focuses on the repair of severely injured skeletal muscle, magnetically stimulated bioagent-free ferrogels may find broad utility in the field of regenerative medicine.

Skeletal muscle atrophy in bioengineered skeletal muscle: a new model system.

Skeletal muscle atrophy has been well characterized in various animal models, and while certain pathways that lead to disuse atrophy and its associated functional deficits have been well studied, available drugs to counteract these deficiencies are limited. An ex vivo tissue-engineered skeletal muscle offers a unique opportunity to study skeletal muscle physiology in a controlled in vitro setting. Primary mouse myoblasts isolated from adult muscle were tissue engineered into bioartificial muscles (BAMs) containing hundreds of aligned postmitotic muscle fibers expressing sarcomeric proteins. When electrically stimulated, BAMs generated measureable active forces within 2-3 days of formation. The maximum isometric tetanic force (Po) increased for ∼3 weeks to 2587±502 µN/BAM and was maintained at this level for greater than 80 days. When BAMs were reduced in length by 25% to 50%, muscle atrophy occurred in as little as 6 days. Length reduction resulted in significant decreases in Po (50.4%), mean myofiber cross-sectional area (21.7%), total protein synthesis rate (22.0%), and noncollagenous protein content (6.9%). No significant changes occurred in either the total metabolic activity or protein degradation rates. This study is the first in vitro demonstration that length reduction alone can induce skeletal muscle atrophy, and establishes a novel in vitro model for the study of skeletal muscle atrophy.

Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system.

Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time-lapse recordings and their subsequent quenching by d-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair.

The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration.

Many cell types of therapeutic interest, including myoblasts, exhibit reduced engraftment if cultured prior to transplantation. This study investigated whether polymeric scaffolds that direct cultured myoblasts to migrate outwards and repopulate the host damaged tissue, in concert with release of angiogenic factors designed to enhance revascularizaton of the regenerating tissue, would enhance the efficacy of this cell therapy and lead to functional muscle regeneration. This was investigated in the context of a severe injury to skeletal muscle tissue involving both myotoxin-mediated direct damage and induction of regional ischemia. Local and sustained release of VEGF and IGF-1 from macroporous scaffolds used to transplant and disperse cultured myogenic cells significantly enhanced their engraftment, limited fibrosis, and accelerated the regenerative process. This resulted in increased muscle mass and, improved contractile function. These results demonstrate the importance of finely controlling the microenvironment of transplanted cells in the treatment of severe muscle damage.

The role of GH and IGF-I in mediating anabolic effects of testosterone on androgen-responsive muscle.

Testosterone (T) supplementation increases skeletal muscle mass, circulating GH, IGF-I, and im IGF-I expression, but the role of GH and IGF-I in mediating T's effects on the skeletal muscle remains poorly understood. Here, we show that T administration increased body weight and the mass of the androgen-dependent levator ani muscle in hypophysectomized as well as castrated plus hypophysectomized adult male rats. T stimulated the proliferation of primary human skeletal muscle cells (hSKMCs) in vitro, an effect blocked by transfecting hSKMCs with small interference RNA targeting human IGF-I receptor (IGF-IR). In differentiation conditions, T promoted the fusion of hSKMCs into larger myotubes, an effect attenuated by small interference RNA targeting human IGF-IR. Notably, MKR mice, which express a dominant negative form of the IGF-IR in skeletal muscle fibers, treated with a GnRH antagonist (acyline) to suppress endogenous T, responded to T administration by an attenuated increase in the levator ani muscle mass. In conclusion, circulating GH and IGF-I are not essential for mediating T's effects on an androgen-responsive skeletal muscle. IGF-I signaling plays an important role in mediating T's effects on skeletal muscle progenitor cell growth and differentiation in vitro. However, IGF-IR signaling in skeletal muscle fibers does not appear to be obligatory for mediating the anabolic effects of T on the mass of androgen-responsive skeletal muscles in mice.

Functional muscle regeneration with combined delivery of angiogenesis and myogenesis factors.

Regenerative efforts typically focus on the delivery of single factors, but it is likely that multiple factors regulating distinct aspects of the regenerative process (e.g., vascularization and stem cell activation) can be used in parallel to affect regeneration of functional tissues. This possibility was addressed in the context of ischemic muscle injury, which typically leads to necrosis and loss of tissue and function. The role of sustained delivery, via injectable gel, of a combination of VEGF to promote angiogenesis and insulin-like growth factor-1 (IGF1) to directly promote muscle regeneration and the return of muscle function in ischemic rodent hindlimbs was investigated. Sustained VEGF delivery alone led to neoangiogenesis in ischemic limbs, with complete return of tissue perfusion to normal levels by 3 weeks, as well as protection from hypoxia and tissue necrosis, leading to an improvement in muscle contractility. Sustained IGF1 delivery alone was found to enhance muscle fiber regeneration and protected cells from apoptosis. However, the combined delivery of VEGF and IGF1 led to parallel angiogenesis, reinnervation, and myogenesis; as satellite cell activation and proliferation was stimulated, cells were protected from apoptosis, the inflammatory response was muted, and highly functional muscle tissue was formed. In contrast, bolus delivery of factors did not have any benefit in terms of neoangiogenesis and perfusion and had minimal effect on muscle regeneration. These results support the utility of simultaneously targeting distinct aspects of the regenerative process.

Tissue engineering skeletal muscle for orthopaedic applications.

With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

Mechanical stimulation improves tissue-engineered human skeletal muscle.

Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.