Skeletal Muscle Constructs Research Articles

Optimisation of cell fate determination for cultivated muscle differentiation.

Production of cultivated meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Although these formulations can drive myogenic differentiation levels comparable to serum-starvation-based protocols, the resulting cultures are often heterogeneous, with a significant proportion of cells not participating in myofusion, limiting maturation of the muscle. To address this problem, we employed RNA sequencing to analyse heterogeneity in differentiating bovine satellite cells at single-nucleus resolution, identifying distinct cellular subpopulations including proliferative cells that fail to exit the cell cycle and quiescent 'reserve cells' that do not commit to myogenic differentiation. Our findings indicate that the MEK/ERK, NOTCH, and RXR pathways are active during the initial stages of myogenic cell fate determination, and by targeting these pathways, we can promote cell cycle exit while reducing reserve cell formation. This optimised medium formulation consistently yields fusion indices close to 100% in 2D culture. Furthermore, we demonstrate that these conditions enhance myotube formation and actomyosin accumulation in 3D bovine skeletal muscle constructs, providing proof of principle for the generation of highly differentiated cultivated muscle with excellent mimicry to traditional muscle.

Current Methodologies for Inducing Aligned Myofibers in Tissue Constructs for Skeletal Muscle Tissue Regeneration.

Significance: Volumetric muscle loss (VML) results in the loss of large amounts of tissue that inhibits muscle regeneration. Existing therapies, such as autologous muscle transfer and physical therapy, are incapable of returning full function and force production to injured muscle. Recent Advances: Skeletal muscle tissue constructs may provide an alternative to existing therapies currently used to treat VML. Unlike autologous muscle transplants, muscle constructs can be cultured in vitro and are not reliant on intact muscle tissue. Skeletal muscle constructs can be generated from small muscle biopsies and could be used to generate skeletal muscle tissue constructs to replace injured tissues. Critical Issues: To serve as effective therapies, muscle constructs must be capable of generating contractile forces that can assist the function of host skeletal muscle. The contractile force of native muscle arises in part as a consequence of the highly aligned, bundled architecture of myofibers. Attempts to induce similar alignment include applications of tension/strain across hydrogels, inducing aligned architectures within scaffolds, casting tissues in straited molds, and 3D printing. While all these methods have demonstrated efficacy toward inducing myofiber alignment, the extent of myofiber alignment, tissue formation, and force production varies. This manusript critically reviews the advantages and limitations of these methods and specifically discusses their ability to impart mechanical and architectural cues to induce alignment within tissue constructs. Future Directions: As tissue-synthesizing techniques continue to improve, muscle constructs must include more cell types than simply myoblasts, such as the addition of neuronal and endothelial cells. Higher-level tissue organization is critical to the success of these constructs. Many of these technologies have yet to be implanted into host tissue to understand engraftment and how they can contribute to traumatic injury, and as such continued collaboration between surgeons and tissue engineers is necessary to ultimately result in clinical translation.

Engineered Regenerative Isolated Peripheral Nerve Interface for Targeted Reinnervation.

A regenerative peripheral nerve interface (RPNI) offers a therapeutic solution for nerve injury through reconstruction of the target muscle. However, implanting a transected peripheral nerve into an autologous skeletal muscle graft in RPNI causes donor-site morbidity, highlighting the need for tissue-engineered skeletal muscle constructs. Here, an engineered regenerative isolated peripheral nerve interface (eRIPEN) is developed using 3D skeletal cell printing combined with direct electrospinning to create a nanofiber membrane envelop for host nerve implantation. In this in vivo study, after over 8 months of RPNI surgery, the eRIPEN exhibits a minimum Feret diameter of 15-20µm with a cross-sectional area of 100-500 µm2, representing the largest distribution of myofibers. Furthermore, neuromuscular junction formation and muscle contraction with a force of ≈28 N are observed. Notably, the decreased hypersensitivity to mechanical/thermal stimuli and an improved tibial functional index from -77 to -56 are found in the eRIPEN group. The present novel concept of eRIPEN paves the way for the utilization and application of tissue-engineered constructs in RPNI, ultimately realizing neuroprosthesis control through synaptic connections.

Generation of Bidimensional and Three-Dimensional Muscle Culture Systems.

Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct.

3D Printed Magneto-Active Microfiber Scaffolds for Remote Stimulation and Guided Organization of 3D In Vitro Skeletal Muscle Models.

This work reports the rational design and fabrication of magneto-active microfiber meshes with controlled hexagonal microstructures via melt electrowriting (MEW) of a magnetized polycaprolactone-based composite. In situ iron oxide nanoparticle deposition on oxidized graphene yields homogeneously dispersed magnetic particles with sizes above 0.5µm and low aspect ratio, preventing cellular internalization and toxicity. With these fillers, homogeneous magnetic composites with high magnetic content (up to 20weight %) are obtained and processed in a solvent-free manner for the first time. MEW of magnetic composites enabled the creation of skeletal muscle-inspired design of hexagonal scaffolds with tunable fiber diameter, reconfigurable modularity, and zonal distribution of magneto-active and nonactive material, with elastic tensile deformability. External magnetic fields below 300mT are sufficient to trigger out-of-plane reversible deformation. In vitro culture of C2C12 myoblasts on three-dimensional (3D) Matrigel/collagen/MEW scaffolds showed that microfibers guided the formation of 3D myotube architectures, and the presence of magnetic particles does not significantly affect viability or differentiation rates after 8 days. Centimeter-sized skeletal muscle constructs allowed for reversible, continued, and dynamic magneto-mechanical stimulation. Overall, these innovative microfiber scaffolds provide magnetically deformable platforms suitable for dynamic culture of skeletal muscle, offering potential for in vitro disease modeling.

Bioengineering approaches to promote maturity in human skeletal muscle constructs

In 2021/22, the MicroAge project, a UK Space Agency-funded mission, sent 3D muscle constructs to the International Space Station to study microgravity as a model of accelerated musculoskeletal ageing on Earth. The aim of this work is to further develop the muscle constructs, compromised of human skeletal muscle cells encapsulated in fibrin hydrogels, induced to differentiate into myotubes and grown in 3D. The study uses several bioengineering approaches to determine the extent of maturity achievable in these muscle constructs in vitro, with the aim of producing a pharmacological or physiological screening platform of mature muscle, targeting muscle disorders such as sarcopenia.To enable measurement of muscle functional properties, it was also necessary to develop a suitable surrogate for force generation due to constraints of strain gauge use, and so this study also determined whether electrochemical impedance spectroscopy (EIS) was appropriate in numerous instances, opening up possibilities for rapid and automated measurement of contractile properties of in vitro skeletal muscle models. This was undertaken by the development of the MicroAge culture system to allow simultaneous measurement of force characteristic using a micro stain gauge and EIS measurements via the stimulating electrodes.The MicroAge muscle constructs have been characterised using a panel of maturation indices for skeletal muscle, including measuring contractile properties, and myosin heavy chain isoform expression using immunocytochemistry with spinning disc microscopy, RT-qPCR and Western blotting and these data will be presented.Of particular interest are the data that demonstrated a linear relationship between force generation measured using the ‘gold-standard’ approach of using a strain gauge and EIS (R2 = 0.9), validating the use of EIS as a proxy for force generation in a range of conditions such as in muscle constructs at rest and during contraction.This allows for rapid and automated measurement of contractile properties of in vitro models of skeletal muscle, providing an exciting new platform to rapidly test pharmacological interventions that may result in improved muscle function in 3D models of disease. The study is also currently implementing bioprinting of muscle constructs to streamline construct formation and investigating varying patterns of electrical stimulation on construct maturity throughout differentiation and the results of this approach will also be presented. Supported by a PhD studentship from MRC DiMeN Doctoral Training Programme and CIMA. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bioengineering approaches to promote maturity in human skeletal muscle constructs.

During ageing, cellular changes impairing normal responses to contractile activity causes our skeletal muscles to atrophy and weaken. Additionally, reduced innervation quality in muscle of older individuals results in denervation atrophy. This declining muscle mass and strength, termed sarcopenia, contributes to frailty and loss of independence in older populations. In order to study sarcopenia pathophysiology, there is a requirement for physiologically relevant in vitro models of mature human skeletal muscle which display neuromuscular junction (NMJ) formation. Recently, the MicroAge project, a UK Space Agency funded mission to the International Space Station, used microgravity to model accelerated musculoskeletal ageing of muscle constructs consisting of human skeletal muscle cells grown in 3D within a hydrogel. This current project aims to explore several bioengineering approaches to promote maturity of these constructs, developing a model of mature human skeletal muscle. The MicroAge constructs will first be characterised against a panel of maturation indices for skeletal muscle, including fibre typing and genetic markers of maturity. Development of a system to simultaneously measure force generation and impedance spectroscopy will evaluate how well impedance spectroscopy monitors tissue maturity as well as being a proxy for force generation during contractions. Subsequently, we aim to implement bioprinting to streamline construct formation, and investigate effects of varying patterns of electrical stimulation on maturity throughout construct differentiation. The longer-term aim is to develop an in vitro NMJ model through innervation of constructs by co-culture with human induced pluripotent stem cells differentiated into neuronal stem cells. Effects of these approaches on construct maturity will be measured by changes to the panel of maturation indices and contractile properties, determining the extent of maturity that can be achieved by human muscle constructs in vitro and producing a platform for screening therapeutic interventions targeting sarcopenia.

Towards innervation of bioengineered muscle constructs: Development of a sustained neurotrophic factor delivery and release system

Surgical implantation of biomanufactured skeletal muscle constructs has recently emerged as a promising strategy to treat volumetric muscle defects. However, due to the slow rate of neural regeneration and integration, timely innervation of the implanted constructs with the host peripheral nerves remains an unresolved challenge. This study aims to develop a sustained release neurotrophic factor (NF) delivery system to accelerate peripheral nerve regeneration and innervation of three-dimensional (3D) bioprinted skeletal muscle constructs. Poly (lactic-co-glycolic acid) (PLGA) microspheres were selected as a delivery system for efficient loading and sustained release of two potent NFs: ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF). We demonstrate that the NFs can be loaded within the PLGA microspheres with a high encapsulation efficiency (75.4% ± 12.6%). The NF-loaded microspheres were incorporated into the fibrinogen-based bioink used to produce biomanufactured skeletal muscle constructs and tested for printability. The microspheres did not change the viscoelastic properties of the bioink, nor did they affect the viability of human muscle progenitor cells. The release kinetic test confirmed that the bioprinted muscle constructs with the NFs-loaded microspheres released the NFs in a sustained manner compared to the bioprinted muscle construct without microspheres. The released NFs maintained their biological activities. In an in vitro neurite outgrowth assay, the NFs released from the PLGA microspheres facilitated the neurite growth over a longer time scale than the NFs directly loaded in the hydrogel. These results demonstrate the feasibility of incorporating the microsphere-based NF delivery system for accelerating neural regeneration in future in vivo applications involving biomanufactured muscle constructs.

Contractile force assessment methods for in vitro skeletal muscle tissues.

Over the last few years, there has been growing interest in measuring the contractile force (CF) of engineered muscle tissues to evaluate their functionality. However, there are still no standards available for selecting the most suitable experimental platform, measuring system, culture protocol, or stimulation patterns. Consequently, the high variability of published data hinders any comparison between different studies. We have identified that cantilever deflection, post deflection, and force transducers are the most commonly used configurations for CF assessment in 2D and 3D models. Additionally, we have discussed the most relevant emerging technologies that would greatly complement CF evaluation with intracellular and localized analysis. This review provides a comprehensive analysis of the most significant advances in CF evaluation and its critical parameters. In order to compare contractile performance across experimental platforms, we have used the specific force (sF, kN/m2), CF normalized to the calculated cross-sectional area (CSA). However, this parameter presents a high variability throughout the different studies, which indicates the need to identify additional parameters and complementary analysis suitable for proper comparison. We propose that future contractility studies in skeletal muscle constructs report detailed information about construct size, contractile area, maturity level, sarcomere length, and, ideally, the tetanus-to-twitch ratio. These studies will hopefully shed light on the relative impact of these variables on muscle force performance of engineered muscle constructs. Prospective advances in muscle tissue engineering, particularly in muscle disease models, will require a joint effort to develop standardized methodologies for assessing CF of engineered muscle tissues.

Assessing Contractility of 3D iPSC‐derived Muscle Models for Safety and Discovery Using a Novel, High‐throughput, and Label‐free Instrumentation Platform

Stem cell models hold great promise for improving the predictive power of preclinical in vitro assays for new therapies, drug discovery, basic scientific research, and disease modeling. Complex 3D platforms, such as Engineered Muscle Tissues (EMTs) fabricated from primary or iPSC‐derived cells, can directly measure tissue contractility, which is challenging in conventional 2D platforms where cells are rigidly attached to a surface. However, traditional methods to fabricate EMTs demand extensive bioengineering expertise, and measuring contractility often involves laborious, serial, and low‐throughput optical measurements.Here, we report on the design, fabrication, and validation of a novel 3D EMT platform that uses 1) facile and scalable bioengineering approaches to generate tissues from a variety of cell sources, and 2) a label‐free parallel measurement technique. Our tissue casting approach has a success rate of >96% (n > 100) and produces consistently‐sized constructs with a standard deviation of +/‐ 9% across 6 experiments. Tissue casting, media changes, and drug dosing are also highly amenable to automation.The substrate features an embedded magnet; as tissues contract, the magnet’s displacement is quantitatively detected in a highly‐parallel manner using specialized sensors. We detected 24 contractions simultaneously with a measurement rate of 100Hz, which is suitable for measuring various aspects of contractility such as upstroke velocity, decay time, and fatigability. We demonstrated that the signal voltage changes are linear with respect to EMT contraction.We will present data showing acute effects of drugs measured minutes after EMT dosing and chronic effects of structural cardiotoxicants like doxorubicin, sunitinib, and BMS‐986094 on EMTs. All chronically‐dosed tissues showed statistically significant dose‐dependent reduction in twitch frequency over a multi‐day time course (p <0.05). We will also show that our platform can be used to generate physiologically‐relevant skeletal muscle constructs and achieve tetanic responses upon stimulation.In addition to modeling healthy tissues, our platform can also be used to study disease models. We are currently developing patient‐specific Duchenne Muscular Dystrophy models for the development of personalized gene therapies. EMTs can be made from cells sourced from patients and used to test whether a new therapy will improve or recover functional contraction.We have designed a novel system that can leverage the complexity of 3D cellular models in a scalable format and can be tailored for specific applications, including personalized medicine or disease modeling. The Mantarray platform will provide a stand‐alone tool capable of screening significant numbers of compounds for the rapid safety evaluation of drug candidates thereby accelerating drug discovery and development.

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects.

Hydrogel-Based Fiber Biofabrication Techniques for Skeletal Muscle Tissue Engineering.

The functional capabilities of skeletal muscle are strongly correlated with its well-arranged microstructure, consisting of parallelly aligned myotubes. In case of extensive muscle loss, the endogenous regenerative capacity is hindered by scar tissue formation, which compromises the native muscle structure, ultimately leading to severe functional impairment. To address such an issue, skeletal muscle tissue engineering (SMTE) attempts to fabricate in vitro bioartificial muscle tissue constructs to assist and accelerate the regeneration process. Due to its dynamic nature, SMTE strategies must employ suitable biomaterials (combined with muscle progenitors) and proper 3D architectures. In light of this, 3D fiber-based strategies are gaining increasing interest for the generation of hydrogel microfibers as advanced skeletal muscle constructs. Indeed, hydrogels possess exceptional biomimetic properties, while the fiber-shaped morphology allows for the creation of geometrical cues to guarantee proper myoblast alignment. In this review, we summarize commonly used hydrogels in SMTE and their main properties, and we discuss the first efforts to engineer hydrogels to guide myoblast anisotropic orientation. Then, we focus on presenting the main hydrogel fiber-based techniques for SMTE, including molding, electrospinning, 3D bioprinting, extrusion, and microfluidic spinning. Furthermore, we describe the effect of external stimulation (i.e., mechanical and electrical) on such constructs and the application of hydrogel fiber-based methods on recapitulating complex skeletal muscle tissue interfaces. Finally, we discuss the future developments in the application of hydrogel microfibers for SMTE.

Fabricating Tissues In Situ with the Controlled Cellular Alignments.

Tissue engineering techniques have enabled to replicate the geometrical architecture of native tissues but usually fail to reproduce their exact cellular arrangements during the fabricating process, while it is critical for manufacturing physiologically relevant tissues. To address this problem, a "sewing-like" method of controlling cellular alignment during the fabricating process is reported here. By integrating the stretching step into the fabricating process, a static mechanical environment is created which, in turn, regulates the subsequent cellular alignment, elongation, and differentiation in the generated tissues. With this method, patterned cellular constructs can be fabricated with controlled cellular alignment. Moreover, this method shows a potent capability to fabricate physiologically relevant skeletal muscle constructs in vitro by mechanically inducing myoblast fusion and maturation. As a potential clinical application, aligned myofibers are directly fabricated onto injured muscles in vivo, which repair the damaged tissues effectively. This study shows that the "sewing-like" method can produce engineered tissues with precise control of cellular arrangements and more clinically viable functionalities.

Multiscale-Engineered Muscle Constructs: PEG Hydrogel Micro-Patterning on an Electrospun PCL Mat Functionalized with Gold Nanoparticles.

The development of new, viable, and functional engineered tissue is a complex and challenging task. Skeletal muscle constructs have specific requirements as cells are sensitive to the stiffness, geometry of the materials, and biological micro-environment. The aim of this study was thus to design and characterize a multi-scale scaffold and to evaluate it regarding the differentiation process of C2C12 skeletal myoblasts. The significance of the work lies in the microfabrication of lines of polyethylene glycol, on poly(ε-caprolactone) nanofiber sheets obtained using the electrospinning process, coated or not with gold nanoparticles to act as a potential substrate for electrical stimulation. The differentiation of C2C12 cells was studied over a period of seven days and quantified through both expression of specific genes, and analysis of the myotubes’ alignment and length using confocal microscopy. We demonstrated that our multiscale bio-construct presented tunable mechanical properties and supported the different stages skeletal muscle, as well as improving the parallel orientation of the myotubes with a variation of less than 15°. These scaffolds showed the ability of sustained myogenic differentiation by enhancing the organization of reconstructed skeletal muscle. Moreover, they may be suitable for applications in mechanical and electrical stimulation to mimic the muscle’s physiological functions.

Contractility and myosin heavy chain content of skeletal muscle engineered from adult and aged rats

Functional three-dimensional skeletal muscle constructs (myooids) were engineered from myogenic cells harvested from the muscles of adult and aged rats. Myooids formed by self-organization of myogenic cells in the absence of an artificial scaffold and were maintained in culture. Myooid excitability and contractility were measured at days 1, 3, 7, and 24 after myooid formation. Skeletal muscle myosin heavy chain (MHC) content was measured and MHC isoforms were separated on SDS-PAGE gels and quantified. Adult and aged rat myooids had only ~35-60% of the skeletal muscle MHC content of control skeletal muscle from rats, the remaining MHC content consisting of isoforms found in cultured fibroblasts but not in control skeletal muscle. In addition, myooids expressed only developmental isoforms of skeletal muscle MHC, known to generate less specific force than adult isoforms. Myooids from aged rats generated greater normalized force than myooids from adult rats, but only 1-3% of that for adult skeletal muscle controls. Normalizing for skeletal muscle MHC content and isoform expression predicts myooid forces of ~6-18% of control adult skeletal muscles. We hypothesize that the remaining force deficit is due to cellular and sub-cellular disorganization—Myooids lack the density and organization of sarcomeric arrays seen in skeletal muscle.

Tissue engineered skeletal muscle model of rheumatoid arthritis using human primary skeletal muscle cells.

Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily targeting the joints. Autoreactive immune cells involved in RA affect other tissues, including skeletal muscle. Patients with RA experience diminished physical function, limited mobility, reduced muscle function, chronic pain, and increased mortality. To explore the impact of RA on skeletal muscle, we engineered electrically responsive, contractile human skeletal muscle constructs (myobundles) using primary skeletal muscle cells isolated from the vastus lateralis muscle of 11 RA patients (aged 57-74) and 10 aged healthy donors (aged 55-76), as well as from the hamstring muscle of six young healthy donors (less than 18years of age) as a benchmark. Since all patients were receiving treatment for the disease, RA disease activity was mild. In 2D culture, RA myoblast purity, growth rate, and senescence were not statistically different than aged controls; however, RA myoblast purity showed greater variance compared to controls. Surprisingly, in 3D culture, contractile force production by RA myobundles was greater compared to aged controls. In support of this finding, assessment of RA myofiber maturation showed increased area of sarcomeric α-actinin (SAA) expression over time compared to aged controls. Furthermore, a linear regression test indicated a positive correlation between SAA protein levels and tetanus force production in RA and controls. Our findings suggest that medications prescribed to RA patients may maintain-or even enhance-muscle function, and this effect is retained and observed in in vitro culture. Future studies regarding the effects of RA therapeutics on RA skeletal muscle, in vivo and in vitro, are warranted.

Decellularized Skeletal Muscles Support the Generation of In Vitro Neuromuscular Tissue Models

Decellularized skeletal muscle (dSkM) constructs have received much attention in recent years due to the versatility of their applications in vitro. In search of adequate in vitro models of the skeletal muscle tissue, the dSkM offers great advantages in terms of the preservation of native-tissue complexity, including three-dimensional organization, the presence of residual signaling molecules within the construct, and their myogenic and neurotrophic abilities. Here, we attempted to develop a 3D model of neuromuscular tissue. To do so, we repopulated rat dSkM with human primary myogenic cells along with murine fibroblasts and we coupled them with organotypic rat spinal cord samples. Such culture conditions not only maintained multiple cell type viability in a long-term experimental setup, but also resulted in functionally active construct capable of contraction. In addition, we have developed a customized culture system which enabled easy access, imaging, and analysis of in vitro engineered co-cultures. This work demonstrates the ability of dSkM to support the development of a contractile 3D in vitro model of neuromuscular tissue fit for long-term experimental evaluations.

A Bioprinting Process Supplemented with In Situ Electrical Stimulation Directly Induces Significant Myotube Formation and Myogenesis

AbstractElectric field stimulation has supported biophysical and biological cues for tissue regeneration approaches to affect cell morphology, alignment, and even cellular phenotypes types. Here, an innovative bioprinting approach supported by in situ electrical stiumlation (E‐printing) is used to fabricate a bioengineered skeletal muscle construct composed of human adipose stem cells and methacrylated decellularized extracellular matrix (dECM‐Ma) derived from porcine muscle. To obtain highly ordered myofiber‐like structures, various parameters of the printing process are optimized. The E‐printed structure exhibits higher cell viability and fully aligned cytoskeleton than the conventionally printed cell‐bearing structures, due to activation of voltage‐gated ion channels that affect various signaling pathways. When using the E‐printed structure, expression of myogenesis‐related genes is upregulated by 1.9–2.5‐fold higher than when using a dECM‐Ma structure produced without electrical stimulation. Furthermore, when implanted into a rat model of volumetric muscle loss, the structure yields outstanding myogenesis relative to the conventionally bioprinted structure.

Replace and repair: Biomimetic bioprinting for effective muscle engineering.

The debilitating effects of muscle damage, either through ischemic injury or volumetric muscle loss (VML), can have significant impacts on patients, and yet there are few effective treatments. This challenge arises when function is degraded due to significant amounts of skeletal muscle loss, beyond the regenerative ability of endogenous repair mechanisms. Currently available surgical interventions for VML are quite invasive and cannot typically restore function adequately. In response to this, many new bioengineering studies implicate 3D bioprinting as a viable option. Bioprinting for VML repair includes three distinct phases: printing and seeding, growth and maturation, and implantation and application. Although this 3D bioprinting technology has existed for several decades, the advent of more advanced and novel printing techniques has brought us closer to clinical applications. Recent studies have overcome previous limitations in diffusion distance with novel microchannel construct architectures and improved myotubule alignment with highly biomimetic nanostructures. These structures may also enhance angiogenic and nervous ingrowth post-implantation, though further research to improve these parameters has been limited. Inclusion of neural cells has also shown to improve myoblast maturation and development of neuromuscular junctions, bringing us one step closer to functional, implantable skeletal muscle constructs. Given the current state of skeletal muscle 3D bioprinting, the most pressing future avenues of research include furthering our understanding of the physical and biochemical mechanisms of myotube development and expanding our control over macroscopic and microscopic construct structures. Further to this, current investigation needs to be expanded from immunocompromised rodent and murine myoblast models to more clinically applicable human cell lines as we move closer to viable therapeutic implementation.

Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands.

Simple SummaryEngineered human skeletal muscle tissue is a platform tool that can help scientists and physicians better understand human physiology, pharmacology, and disease modeling. Over the past few years this area of research has been actively being pursued by many labs worldwide. Significant challenges remain, including accessing an adequate cell source, and achieving proper physiological-like architecture of the engineered tissue. To address cell resourcing we aimed at further optimizing a process called transdifferentiation which involves the direct conversion of fibroblasts into skeletal muscle cells. The opportunity here is that fibroblasts are readily available and can be expanded sufficiently to meet the needs of a tissue engineering approach. Additionally, we aimed to demonstrate the applicability of transdifferentiation in assembling tissue engineered skeletal muscle. We implemented a screening process of protein ligands in an effort to refine transdifferentiation, and identified that most proteins resulted in a deficit in transdifferentiation efficiency, although one resulted in robust expansion of cultured cells. We were also successful in assembling engineered constructs consisting of transdifferentiated cells. Future directives involve demonstrating that the engineered tissues are capable of contractile and functional activity, and pursuit of optimizing factors such as electrical and chemical exposure, towards achieving physiological parameters observed in human muscle.The development of robust skeletal muscle models has been challenging due to the partial recapitulation of human physiology and architecture. Reliable and innovative 3D skeletal muscle models recently described offer an alternative that more accurately captures the in vivo environment but require an abundant cell source. Direct reprogramming or transdifferentiation has been considered as an alternative. Recent reports have provided evidence for significant improvements in the efficiency of derivation of human skeletal myotubes from human fibroblasts. Herein we aimed at improving the transdifferentiation process of human fibroblasts (tHFs), in addition to the differentiation of murine skeletal myoblasts (C2C12), and the differentiation of primary human skeletal myoblasts (HSkM). Differentiating or transdifferentiating cells were exposed to single or combinations of biological ligands, including Follistatin, GDF8, FGF2, GDF11, GDF15, hGH, TMSB4X, BMP4, BMP7, IL6, and TNF-α. These were selected for their critical roles in myogenesis and regeneration. C2C12 and tHFs displayed significant differentiation deficits when exposed to FGF2, BMP4, BMP7, and TNF-α, while proliferation was significantly enhanced by FGF2. When exposed to combinations of ligands, we observed consistent deficit differentiation when TNF-α was included. Finally, our direct reprogramming technique allowed for the assembly of elongated, cross-striated, and aligned tHFs within tissue-engineered 3D skeletal muscle constructs. In conclusion, we describe an efficient system to transdifferentiate human fibroblasts into myogenic cells and a platform for the generation of tissue-engineered constructs. Future directions will involve the evaluation of the functional characteristics of these engineered tissues.