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.

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.

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.

Minimally invasive approach to the repair of injured skeletal muscle with a shape-memory scaffold.

Repair of injured skeletal muscle by cell therapies has been limited by poor survival of injected cells. Use of a carrier scaffold delivering cells locally, may enhance in vivo cell survival, and promote skeletal muscle regeneration. Biomaterial scaffolds are often implanted into muscle tissue through invasive surgeries, which can result in trauma that delays healing. Minimally invasive approaches to scaffold implantation are thought to minimize these adverse effects. This hypothesis was addressed in the context of a severe mouse skeletal muscle injury model. A degradable, shape-memory alginate scaffold that was highly porous and compressible was delivered by minimally invasive surgical techniques to injured tibialis anterior muscle. The scaffold controlled was quickly rehydrated in situ with autologous myoblasts and growth factors (either insulin-like growth factor-1 (IGF-1) alone or IGF-1 with vascular endothelial growth factor (VEGF)). The implanted scaffolds delivering myoblasts and IGF-1 significantly reduced scar formation, enhanced cell engraftment, and improved muscle contractile function. The addition of VEGF to the scaffold further improved functional recovery likely through increased angiogenesis. Thus, the delivery of myoblasts and dual local release of VEGF and IGF-1 from degradable scaffolds implanted through a minimally invasive procedure effectively promoted the functional regeneration of injured skeletal muscle.

Sustained delivery of VEGF maintains innervation and promotes reperfusion in ischemic skeletal muscles via NGF/GDNF signaling.

Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.

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.

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.

Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts.

Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96-microwell plates into 3-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty-one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin-like growth factor-1, creatine, beta-hydroxy-beta-methylbutyrate, trichostatin A, lisinopril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC(50) of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders.

Sensorial and Nutritional Aspects of Cultured Meat in Comparison to Traditional Meat: Much to Be Inferred.

Cultured meat aspires to be biologically equivalent to traditional meat. If cultured meat is to be consumed, sensorial (texture, color, flavor) and nutritional characteristics are of utmost importance. This paper compares cultured meat to traditional meat from a tissue engineering and meat technological point of view, focusing on several molecular, technological and sensorial attributes. We outline the challenges and future steps to be taken for cultured meat to mimic traditional meat as closely as possible.

Cellular strain assessment tool (CSAT): precision-controlled cyclic uniaxial tensile loading.

The development of a multi-sample strain device and elastomeric culture wells designed to systematically assess strain effects on cell cultures is presented in this report. This device enables one to precisely conduct experimental analyses in sterile conditions while delivering cyclic uniaxial tensile strain. The input to the computer interface allows one to alter variables of frequency, duration, and amplitude of strain. The influence of strain on the migration of human umbilical vein endothelial cell (HUVEC) cultured on 2D polydimethylsiloxane (PDMS) surfaces was examined to verify the utility of this system.

Coculture Method to Obtain Endothelial Networks Within Human Tissue-Engineered Skeletal Muscle.

Skeletal muscle tissue engineering aims at creating functional skeletal muscle in vitro. Human muscle organoids can be used for potential applications in regenerative medicine, but also as an in vitro model for myogenesis or myopathology. However, the thickness of constructs is limited due to passive diffusion of nutrients and oxygen. Introduction of a vascular network in vitro may solve this limitation. Here, we describe tissue engineering of in vitro skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. To create bio-artificial muscle (BAM), human muscle progenitor cells are cocultured with human umbilical vein endothelial cells (HUVECs) in a fibrin hydrogel. The cell-gel mix is cast into silicone molds with end attachment sites and cultured in endothelial growth medium (EGM-2) for 1 week. The passive forces generated in the contracted hydrogel align the myogenic cells parallel to the long axis of the contracted gel such that they fuse into aligned multinucleated myofibers. This results in the formation of a 2 cm long and ~1.5 mm tick human BAM construct with endothelial networks.

Growth, differentiation, transplantation and survival of human skeletal myofibers on biodegradable scaffolds.

Skeletal muscle transplantation strategies for muscle repair or gene therapy involve either the injection of proliferating myoblasts followed by fusion with host myofibers or implantation of ex vivo differentiated myofibers; however, both implant procedures are associated with significant cell loss. Biodegradable porous, gas-foamed poly-lactide-co-glycolide (PLG) scaffolds have desirable characteristics for cell transfer and were used to study attachment, growth, differentiation and survival of human myogenic cells. Primary human myoblasts suspended in clinical grade extracellular matrixes (ECMs) and adhered to PLG scaffolds differentiated in vitro into high-density tropomyosin positive myofibers. An immunodeficient non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse implant model was used to study the transfer and in vivo survival of differentiated human myofibers on these scaffolds. Scaffold rigidity allowed the myofibers to be maintained under tension in vitro and following subcutaneous transplantation in vivo. Following implantation, myofiber density on the PLG scaffolds decreased linearly by 78% over a 4-week period. ECM composed of either Tisseel fibrin or Zyderm collagen type I did not significantly affect in vivo cell viability over the 4-week period. Varying PLG scaffold microsphere content (10-100%) also had little effect on cell survival in vivo. In contrast, when the residual NK cell population in the immunodeficient NOD/SCID mouse model was depleted with anti-asialo GM1 (ASGM1) antiserum, in vivo cell survival significantly increased from 22% to 34% after 4 weeks. With further improvements in cell survival, PLG scaffolds may prove useful for the implantation of primary human myofibers in future clinical applications.

Engineering of Human Skeletal Muscle With an Autologous Deposited Extracellular Matrix.

Adult skeletal muscle progenitor cells can be embedded in an extracellular matrix (ECM) and tissue-engineered to form bio-artificial muscles (BAMs), composed of aligned post-mitotic myofibers. The ECM proteins which have been used most commonly are collagen type I and fibrin. Fibrin allows for in vitro vasculogenesis, however, high concentrations of fibrinolysis inhibitors are needed to inhibit degradation of the ECM and subsequent loss of BAM tissue structure. For in vivo implantation, fibrinolysis inhibition may prove difficult or even harmful to the host. Therefore, we adapted in vitro culture conditions to enhance the deposition of de novo synthesized collagen type I gradually replacing the degrading fibrin ECM. The in vitro viscoelastic properties of the fibrin BAMs and deposition of collagen were characterized. BAMs engineered with the addition of proline, hydroxyproline, and ascorbic acid in the tissue culture medium had a twofold increase in Young's Modulus, a 2.5-fold decrease in maximum strain, and a 1.6-fold increase in collagen deposition. Lowering the fibrin content of the BAMs also increased Young's Modulus, decreased maximum strain, and increased collagen deposition. Tissue engineering of BAMs with autologous ECM may allow for prolonged in vivo survival.

Improved magnetic regulation of delivery profiles from ferrogels.

While providing the ability to magnetically enhance delivery rates, ferrogels have not been able to produce the various types of regulated delivery profiles likely needed to direct complex biological processes. For example, magnetically triggered release after prolonged periods of payload retention have not been demonstrated and little has been accomplished towards remotely controlling release rate through alterations in the magnetic signal. Also, strategies do not exist for magnetically coordinating multi-drug sequences. The purpose of this study was to develop these capabilities through improved ferrogel design and investigating how alterations in the magnetic signal impact release characteristics. Results show that delivery rate can be remotely regulated using the frequency of magnetic stimulation. When using an optimized biphasic ferrogel design, stimulation at optimized frequencies enabled magnetically triggered deliveries after a delay of 5 days that were 690- to 1950-fold higher than unstimulated baseline values. Also, a sequence of two payloads was produced by allowing one payload to initially diffuse out of the ferrogel, followed by magnetically triggered release of a different payload on day 5. Finally, it was demonstrated that two payloads could be sequentially triggered for release by first stimulating at a frequency tuned to preferentially release one payload (after 24 h), followed by stimulation at a different frequency tuned to preferentially release the other payload (After 4 days). The strategies developed here may expand the utility of ferrogels in clinical scenarios where the timing and sequence of biological events can be tuned to optimize therapeutic outcome.

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.

Paracrine release of insulin-like growth factor 1 from a bioengineered tissue stimulates skeletal muscle growth in vitro.

Bioengineered tissues transduced to secrete recombinant proteins may serve as a long-term delivery vehicle for therapeutic proteins when implanted in vivo. Insulin-like growth factor 1 (IGF1) is an anabolic growth factor for skeletal muscle that can stimulate myoblast proliferation and myofiber hypertrophy. To determine whether the release of IGF1 from an engineered bioartificial skeletal muscle (BAM) could stimulate the growth of skeletal muscle in a paracrine manner, we established an in vitro perfusion system for genetically engineered IGF1 BAMs. BAMs were bioengineered from C2C12 murine myoblasts stably transduced with a retroviral vector to synthesize and secrete IGF1 (C2-IGF1 BAMs). C2-IGF1 BAMs or nontransduced control C2 BAMs were cocultured with avian BAMS (ABAMs) in constantly perfused biochambers. During 11 days of perfusion, IGF1 levels in the C2-IGF1 BAM perfusion medium increased linearly from 1 to 20 ng/mL. The ABAMs maintained in biochambers with the C2-IGF1 BAMs had significantly more myofibers (69%, p < 0.005) and larger myofiber cross-sectional areas (40%, p < 0.001) compared to those cocultured with control C2 BAMs. These studies show that levels of IGF1 secreted from the C2-IGF1 BAMs are sufficient to produce an anabolic paracrine effect on nongenetically engineered BAMs, and the in vitro perfusion system provides a model for screening proteins effective in stimulating localized skeletal muscle growth.

Sequential release of nanoparticle payloads from ultrasonically burstable capsules.

In many biomedical contexts ranging from chemotherapy to tissue engineering, it is beneficial to sequentially present bioactive payloads. Explicit control over the timing and dose of these presentations is highly desirable. Here, we present a capsule-based delivery system capable of rapidly releasing multiple payloads in response to ultrasonic signals. In vitro, these alginate capsules exhibited excellent payload retention for up to 1 week when unstimulated and delivered their entire payloads when ultrasonically stimulated for 10-100 s. Shorter exposures (10 s) were required to trigger delivery from capsules embedded in hydrogels placed in a tissue model and did not result in tissue heating or death of encapsulated cells. Different types of capsules were tuned to rupture in response to different ultrasonic stimuli, thus permitting the sequential, on-demand delivery of nanoparticle payloads. As a proof of concept, gold nanoparticles were decorated with bone morphogenetic protein-2 to demonstrate the potential bioactivity of nanoparticle payloads. These nanoparticles were not cytotoxic and induced an osteogenic response in mouse mesenchymal stem cells. This system may enable researchers and physicians to remotely regulate the timing, dose, and sequence of drug delivery on-demand, with a wide range of clinical applications ranging from tissue engineering to cancer treatment.

Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2 × 10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1 × 10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2 × 10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.

In vitro Differentiation of Functional Human Skeletal Myotubes in a Defined System.

In vitro human skeletal muscle systems are valuable tools for the study of human muscular development, disease and treatment. However, published in vitro human muscle systems have so far only demonstrated limited differentiation capacities. Advanced differentiation features such as cross-striations and contractility have only been observed in co-cultures with motoneurons. Furthermore, it is commonly regarded that cultured human myotubes do not spontaneously contract, and any contraction has been considered to originate from innervation. This study developed a serum-free culture system in which human skeletal myotubes demonstrated advanced differentiation. Characterization by immunocytochemistry, electrophysiology and analysis of contractile function revealed these major features: A) well defined sarcomeric development, as demonstrated by the presence of cross-striations. B) finely developed excitation-contraction coupling apparatus characterized by the close apposition of dihydropyridine receptors on T-tubules and Ryanodine receptors on sarcoplasmic reticulum membranes. C) spontaneous and electrically controlled contractility. This report not only demonstrates an improved level of differentiation of cultured human skeletal myotubes, but also provides the first published evidence that such myotubes are capable of spontaneous contraction. Use of this functional in vitro human skeletal muscle system would advance studies concerning human skeletal muscle development and physiology, as well as muscle-related disease and therapy.