Engineered muscle tissues for disease modeling and drug screening applications

aDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada bInstitute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada cGraduate School of Environmental Studies, Tohoku University, Sendai, Japan dBiosensors for Bioengineering group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 15-21, 08028 Barcelona, Spain


Introduction
It is obvious that harmonized movement is one of the most significant and common features of humans, which occurs by utilizing their muscles.Muscle tissues are made up of cells with the ability to change their length through contractile process.Skeletal muscle and cardiac muscle are the most important groups of vertebrates' muscles.A muscle that sticks to a bone of the skeleton and is responsible for preserving position and moving the body is called skeletal muscle and a heart muscle is named cardiac muscle.Microscopic images of skeletal and cardiac muscle cells (striated muscle) revealed a series of repeating units called sarcomeres, whereas smooth muscle cells lack this characteristic (1,2).Cardiomyocytes (CMs), endothelial cells (ECs), and cardiac fibroblasts constitute the native myocardium (3).The synchronous contraction of heart occurs due to CMs, which take up almost 90% of the myocardium volume (4).Extracellular matrix (ECM) surrounds CMs and provides physical support and biochemical cues to the cells (5).In particular, mitochondria supply continuous adenosine triphosphate that is needed in maintaining the cardiac contraction.CMs are connected end to end by intercalated disk junctions to form cardiac muscle (Figure 1-A).Besides overall resemblance between skeletal and cardiac tissues, there are some differences between them.Human skeletal muscle exhibits higher degree of multi-nucleation and a cardiac muscle.Skeletal muscles contract in response to a specific stimulus conveyed via a neuro-muscular junction and can be controlled at any time, whereas cardiac muscle exhibits autonomous activity and spontaneous contractions in cell culture (1).A heart muscle has to contract over 100,000 times a day without any break and relaxation (based on ordinary 70 beats per minutes) (6). .Skeletal muscles, constituting about 40 percent of our body's weight, are made up of long cylindrical cells containing many nuclei, which are skeletal muscle fibers.The length and diameter of fibers differ from a few millimeters to more than 10 cm and 5 to 10 µm, respectively.Despite their long length, only few fibers reach the length of muscle tissue..By magnifying the muscle fiber we reach myofibrils, which are also called myotubes (filamentary bundles with the approximate diameter of 0.1 µm, Figure 1-B) (7).
Myofibers are made of a large number of sarcomeres that connect end to end with each other.Sarcomeres, as essential contractile units in striated muscles are separated from adjacent ones using Z-line (a thin border line between sarcomeres).The contractile process of skeletal muscle does not occur without interaction between two fundamental proteins in sarcomeres (i.e., myosin and actin) (1).One way to characterize a mature skeletal muscle is the analysis of sarcomere structure and contraction ability of muscles (8).
Cell survival, motility, and communication take place inside the ECM, which fills spaces among cells with specific biomolecules.In addition to provide mechanical structure for cells, the ECM is a suitable milieu for cell-cell and cell-matrix interactions, migration, differentiation, elongation, and proliferation of cells (9).The ECM for skeletal muscle is mainly composed of collagen type I with minor quantities of collagen types III, IV and V, elastin, fibronectin, and laminin (10).In the other hand, the ECM for cardiac muscle mostly contains collagen types I and III, elastin, and glycosaminoglycans (11).
In what follows, we first describe the most important fabrication methods to make functional muscle tissue constructs.Current applications of engineered muscle tissues in disease modeling and drug screening are then reviewed and discussed.

Engineering muscle tissues
Cardiovascular diseases (CVDs) (e.g., myocardial infarction (12)) are among the most life-threatening disorders world-wide.According to the American Heart Association, CVDs are the leading cause of death internationally (13).It is anticipated that by 2030, 23.6 million people will die because of CVDs, such as heart failure, arrhythmia, and heart valve problems.CVD-related parameters may affect cholesterol, fat, and insulin levels in the body (14).In addition, loss of skeletal muscle functionality is inevitable due to muscle diseases, aging, and injuries.Due to the paucity of allograft muscle tissues for transplantation, muscle tissue engineering as a promising strategy has drawn attention to provide alternative methods to overcome muscle diseases and injuries.Tissue engineering, with the ultimate goal of developing functional tissues, aims to recover or replace damaged or diseased tissues and organs (2).As a critical point in tissue engineering, designing and fabricating a suitable microenvironment for cells that resembles the natural ECM is playing an important role.Scaffolds as the biomimetic ECM should have structural, mechanical, and biological properties close to those in the ECM.In addition, they need to be biocompatible and biodegradable.It is considered that reproducing physico-chemical properties of each specific native tissue environment provides ideal design criteria for tissue engineering scaffolds (15).Microfabrication techniques have been utilized in fabricating functional tissue constructs during the past decades.Here, we highlight commonly used technologies (i.e., electrospinning, bioprinting, dielectrophoresis (DEP), textile approaches, and microfluidics) in manipulating muscle cells and making functional muscle tissues.
Electrospinning is a simple, low-cost, and adaptable approach for synthesizing polymeric fibers from a polymer melt or solution using an electric field.The size of fibers (ranging from a few nanometers (nanofibers) to microns) can be controlled by some factors, such as solution feed rate, applied voltage, and viscosity of polymeric solution (Figure 2-A) (2,25,26).The main reason to utilize the electrospinning technique for tissue engineering applications is morphological resemblance between electrospun fibers as the scaffold and natural ECM, such as Type I collagen (2,27).Besides cell attachment and proliferation, desirable oxygen and nutrient transport properties can also be achieved as a result of high surface area/volume ratio and microporosity of electrospun fibers (28,29).By choosing appropriate material or composite for electrospun fibers, it is possible to tune proper geometries and properties of fiber scaffolds (30).
Precise control of fiber alignment is a key parameter for application of electrospun fibers in muscle tissue engineering.Scaffolds with anisotropic or unidirectional orientation guide morphogenesis of muscle cells and boost cell functionality compared to randomly oriented fibers.Choi et al. used a mixture of polycaprolactone (PCL) and collagen as electrospun nanofiber scaffolds and seeded human skeletal muscle cells on them.Their results showed myotube length on the unidirectional oriented scaffolds was more than twice of that observed on randomly aligned ones, whereas no noticeable differences were reported between myotube diameters (31).In another study, poly(lactic-co-glycolic acid) was employed to fabricate highly aligned electrospun fibers with a diameter ranging from 100-nm to 1.4-µm.The synthesized scaffolds without any surface modification provided appropriate conditions for cell attachment, alignment, differentiation, and proliferation of C2C12 murine myoblasts (26).
Applying electrical stimulation to electrospun fibers (for example PCL or polyurethane (PU) fibers) can improve tissue formation and functionality.Adding multiwalled carbon nanotubes (CNTs) in electrospun fibers also induced muscle cell differentiation and increased electrical conductivity of fibers (32).Electrospun CNT-PU scaffolds enhanced differentiation of myoblasts to myotubes under electrical stimulation compared with the same scaffolds without stimulation (15).Liao et al. studied the effect of electrical stimulation on muscle cells cultured on aligned PU electrospun fibers.Their results showed an increase from 70% to 85% of striated myotubes after applying electrical stimulation on day 7 of culture.Higher cellular elongation and alignment and more striated myotubes were also observed on aligned fibers compared with random fibers (8).
Besides widespread applications of the electrospinning technique, there are few practical limitations (2).For example, as fiber diameter decreases to the nanoscale, the average pore size of the scaffold and consequently cellular infiltration into the fibers decreases.This results in non-uniform dispersion and migration of cells onto the electrospun nanofibers that restrict vascularization and tissue formation (33).Moreover, a potential source of cytotoxicity are chemical residues from electrospun fibers.It is suggested to utilize water as a solvent in order to prevent such toxicity (34).Insufficient mechanical strength is a challenge for some electrospun scaffolds, which is necessary for load bearing applications and appropriate biodegradation of fibers (35).Finally, slow rate of production is an issue that discourages people from using the electrospinning technique (36).

Bioprinting
In 1986 Charles W. Hull called his three-dimensional (3D) printing method "stereolithography" for the first time.He utilized ultraviolet light to cure consecutively printed thin layers of material to make a 3D construct (37).This technology is able to rapidly fabricate micro-and macroarchitectures without compromising the cellular viability (38).A 3D tissue construct can be formed from precise placement of biomaterials and living cells sequentially using the 3D printing approach.The main bioprinting methods include inkjet bioprinting (39)(40)(41), laser-assisted bioprinting (LAB) (42)(43)(44), and extrusion-based bioprinting (EBB) (45)(46)(47).Inkjet bioprinting method is the most prevalent one, which utilizes thermal (39) or acoustic (48) forces to expel desirable amounts of materials on the anticipated location.However, liquids can only exit from inkjet bioprinters and need further treatment to form solids. LAB is based on laserinduced forward transfer in which metals, peptides, DNA, and cells can be printed (43).
Unlike other methods, LAB is nozzle-free and enables printing of materials with a wide range of viscosity and cells with high cell viability and functionality.Despite its advantages, slow flow rate and high setup cost are the major concerns associated with using this technique (49).EBB is appreciated for its notable deposition and printing speed, affordable and commercialized hardware, and ability to print a wide range of bioinks, such as cell encapsulated hydrogels (50), microcarriers (51), and cell aggregates (52).
Compared to the aforementioned methods, limited resolution, solidification process, and low cell viability due to shear stress of the nozzle are major disadvantages of EBB (53).
Bioprinting is an outstanding technique to spatially pattern and distribute cells and ligands inside scaffolds.Kang et al. recently introduced an integrated tissue-organ printer for creation of tissue structures (Figure 2-B).They printed mouse myoblasts to form 3D muscle structures (15 mm × 5 mm × 1 mm).At day 3 of culture, linear stretching and compaction of viable cells were observed and the formation of muscle-like constructs having aligned myotubes was reported at day 7 of culture.After 2 weeks of implantation, nerve integration and vascularization were revealed by the expression of specific markers (54).In another study, differentiation of C2C12 muscle cells incorporating chemical and geometric cues was investigated.An inkjet bioprinter was utilized in printing bone morphogenetic protein-2 and fibroblast growth factor-2 to induce the differentiation of mouse C2C12 myoblasts into tenocytes and osteoblasts, while in the absence of growth factors myotubes were formed (55).

Dielectrophoresis
DEP is an electrokinetic technique applied for cellular characterization (56), particle separation (57,58) and manipulation (59)(60)(61), and fabrication of biomimetic scaffolds (62,63).This technique was first introduced by Herbert Pohl in the 1950s (64).DEP is defined as the motion of dielectric particles within a medium due to an exposed inhomogeneous electric field (65,66).Migration of particles occurs as a result of an electric field gradient in medium.An AC field is usually used to induce the polarization of particles and medium.
The DEP force is influenced by particles' dielectric behavior and size, frequency of electric field and electrical characteristics of medium (67).If the particle is more polarizable compared to the occupying medium, the particle is driven to areas with large electric fields.This occurrence is noted as positive DEP.On the other hand, negative DEP refers to the case where the particle is less polarizable in comparison to the medium.This leads to migration of particles from regions of high electric field (68).
DEP was employed for aligning nanoparticles in scaffolds for tuning their characteristics for tissue regeneration and cell therapy applications (69).For example, we successfully fabricated functional skeletal muscle tissues using dielectrophoretically aligned CNThydrogel scaffolds (70).The CNTs were aligned in gelatin methacrylate (GelMA) hydrogel via DEP for controlling electrical and mechanical properties of scaffolds (Figure 2-C).The Young's modulus of the resulted hydrogel was 23.4 ± 0.2 kPa (close to the elasticity of the native muscle tissue).In addition, the hydrogel showed anisotropic electrical conductivity for fabrication of aligned and contractile muscle myofibers (71).
Different factors, such as voltage, frequency, and ionic conductivity of medium can influence properties of dielectrophoretically manipulated scaffolds (72).Dunne et al. explored effects of some parameters on properties of silk nanofibrous scaffolds prepared by the DEP technique.They reported that decrease in the fibril size can be achieved by increasing the DEP frequency (62).However, there are some limitations and disadvantages associated with the DEP approach.For example, dynamic particles have altering dielectric characteristics based on their size and shape.Therefore, particles of the same type may have different dielectric properties in DEP devices (66).In addition, DEP needs precise sample handling in small amounts, which limits its application in large scale (73).

Textile technology
Textile technologies, originally developed for manufacturing clothes and ornamental fabrics, have newly become a promising approach for fabrication of fibrous scaffolds in tissue engineering (74)(75)(76).The variety of textile manipulation approaches (e.g., weaving (77,78), knitting (79,80), and braiding (81,82) provide accurate control over pore size, surface topography, and pore interconnectivity of fibers and cellular distribution within them (83).Constituent units of textile fibers in tissue engineering can be natural (84), synthetic (85,86), and composite (87,88) fibers.The Acorn CorCap TM cardiac support device is an example of a textile based device used in the cardiac field (89).In addition to mechanical property, each device or graft provides cellular attachment, proliferation, and alignment.Therefore, textile-based grafts and devices can be specified for different tissues or organs (77).Some research groups have utilized textile techniques in cardiac tissue engineering (90).This fabrication method can provide suitable mechanical properties and cellular alignment for CMs.As an outstanding example, knitted mesh of neonatal rat heart cells and fibrin formed a hybrid cardiac construct (91).Hyaluronan benzyl ester was selected as a knit, since hyaluronan is available in the native cardiac tissue.The presence of knit enhanced the mechanical properties of constructs, which experienced cyclic mechanical load in the experiment.This hybrid cardiac construct revealed higher modulus and tensile strength than the native myocardium.Other fabrication methods can be incorporated with textiles to achieve desirable characteristics of scaffolds.Şenel-Ayaz et al. utilized both electrospinning and textile technology to create textile-template electrospun fibers showing various 3D micropatterns based on the type of fabric used as a template (83).In addition, heart valve engineering has also been performed based on textile and electrospinning methods (92,93).By incorporating micromolding technique with textile technology, Hosseini et al. introduced a simple approach, called fiber assisted molding (FAM) for making curved micropatterns (94).They utilized FAM to fabricate 3D helical structures to achieve highly aligned myoblasts and myotubes.

Microfluidics
Microfluidics is a technology that is generally defined by perfusion of fluids in channels with miniaturized diameters ranging from tens to hundreds of micrometers (95).
Microfluidic systems reduce sample volume compared to macroscale counterparts and thereby decrease reagent cost (96).Microfluidic techniques gain advantages from specific features of the microscale world, such as laminar flow that prevents fluid mixing in the microchannels (97).Besides surface tension, capillary force is a predominant factor that should be considered in the design of microfluidic platforms (98).Microfluidics is an interdisciplinary field that has a wide range of applications in biomedical engineering, chemical biosensors, cell analysis, and diagnostics (99).For instance, lab-on-a-chip concept that has attracted worldwide attention in the last decade is mainly relies on microfluidics (100).
In addition to heart-on-chip platforms, microfluidic technology is utilized for many biomedical applications including bio-actuators (101), contraction analysis of cells (102), and dynamic cell culture models (103).Shimizu et al. fabricated a microfluidic device to achieve 3D contractile skeletal muscle tissues.They used C2C12 cells embedded inside collagen and made gels inside microchannels.The muscle contraction was observed under different electrical stimulation conditions (twitch (1 Hz) and tetanus (50 Hz)).
In addition to disease modeling and drug screening, engineered muscle tissues may have other applications in regenerative medicine (104), bio-actuators or bio-robots (105), biological electrochemical systems (gaining electricity and other source of energy from muscle contraction) (106), and food industry (107).These important applications are not the scope of this review.Therefore, in the next section, we focus on the applications of engineered muscle tissues in disease modeling and drug screening.

Applications of engineered muscle tissues in drug screening
Most studies for drug development use animals to model pharmacological response of the human body.Physiological and metabolic differences between animals and humans make it difficult to precisely model the effect of drugs.Recent advances in biomaterial development, tissue engineering, and microscale technologies have helped to develop functional organs-on-a-chip platforms for different drug screening and diagnostics applications.However, despite significant advances in the field of tissue engineering, little work has been done towards using 3D engineered tissues as functional tools in pharmacological research and development.Likewise, little investigations have been devoted to using 3D muscle tissues in pharmacological applications.
In vitro studies should mimic the natural response of human tissues in laboratory.To achieve this goal, a culture system should be constructed according to the physiology of healthy or diseased tissue or organ.Generally, several microenvironment cues should be provided for muscle cells to enhance their maturation and functionality in vitro (108).For example, growth factors and other supplements have been used as the biological cues to regulate muscle cell behavior (109).Insulin-like growth factor 1 (110) and transforming growth factor beta 1 (111) are among the most important growth factors that enhance the maturation and contractility of muscle cells.In addition, substrate properties play an important role in proliferation and differentiation of muscle cells by providing elastic substrates, similar to the native niche (112).3D cell cultures barricade improper signaling and provide better cell-cell and cell-ECM communications compared to 2D platforms (113).Mechanical stretch regimes (passive tension or active forces) revealed potential capability to elevate the maturation and differentiation of different cell types especially muscle cells.For instance, Montevecchi et al. seeded C2C12 cells on electrospun membranes (114).Their results showed that by applying cyclic stretching (three successive movement with frequency of 0.5 Hz, which induced 3.4% deformation of substrate), myosin accumulation was increased eight times compared to non-stimulated cultures.Besides applying mechanical cues to enhance cell differentiation, alignment, and functionality (115), electrical stimulation can exercise muscle cells and persuade cells to obtain and improve their structural and functional ability (116).We electrically stimulated myotubes through interdigitated array of Pt electrodes deposited under the muscle tissue.
Electrical stimulation of C2C12 myotubes (voltage and frequency were 6 V and 1 Hz) increased the alignment and elongation of myotubes (Figure 3-A, B) (117).
There are other factors that need to be considered for mimicking the natural environment of muscle cells, such as cell alignment (118).In Figure 3-C, electrical and topographical cues are incorporated to achieve aligned myotubes (71).Moreover, co-culturing of muscle cells with appropriate surrounding cells provides an in vivo-like environment that enhances the cellular maturation.For instance, co-culturing skeletal muscle with motor neurons, endothelial cells or tenocytes has shown a significant impact on muscle tissue maturation (119).Recently, making neuromuscular junctions in engineered skeletal muscle tissues has gained an increasing attention (120,121).Tissue development in vivo and in vitro are time dependent (122).In the other word, by passing more time muscle cells become more mature.Smith et al. showed that contractile force of myotubes had a noticeable increase from day 14 to day 21 in contrast to first two weeks of culture (123).

Cardiac muscle
Significant advances have been made in the field for cardiac regeneration (124)(125)(126)(127).Some regeneration strategies focus on scaffold-free concepts, such as the generation of multi-layered cardiac cell sheets (128)(129)(130)(131) and the generation of multicellular aggregates named spheroids (132,133).On the other hand, scaffold-based approaches use both natural (134)(135)(136)(137)(138)(139)(140) and synthetic (141-144) biomaterials as the scaffold.Conventional twodimensional (2D) platforms for drug testing are static and do not mimic the complicated cell-matrix and cell-cell interactions in the native cardiac tissue (145,146).The combination of tissue engineering with microfluidic technologies has expedited the development of heart-on-a-chip platforms that can be used as emerging tools for in vivolike applications of cardiac tissues (147)(148)(149).For example, Tanaka et al. developed a novel microspherical heart-like pump device powered by spontaneously contracting neonatal rat CM sheets (101).This system was fabricated by rolling a beating CM sheet onto a fabricated polydimethylsiloxane (PDMS) hollow elastomeric sphere fixed with inlet and outlet ports.Young et al. fabricated a 2-layer microfluidic device to study the permeability of EC monolayers on porous membranes under shear stress conditions (150).
The PDMS device consisted of lower and upper microchannels and a porous membrane separated them.Later, the authors modified the microfluidic device to recapitulate 3D vascular system and studied the physiological cardiovascular cell-cell interactions (151).
Li et al. fabricated a microfluidic chip integrated with an acoustic wave sensor to study the cardiac muscle cell contraction (152).The device has potential application to study contractile activity of a single heart muscle cell during drug screening.
Recent studies have been devoted to developing heart-on-a-chip platforms for pharmaceutical applications (153)(154)(155)(156)(157)(158).Earlier studies used CMs derived from animals, as they were widely accessible.For example, Parker and colleagues developed muscular thin film (MTF)-based systems and employed them for pharmacological and contractility studies (159)(160)(161).They quantified the contractility of rat CMs as a result of epinephrine Recently, Kaneko et al. developed a cell-based model using an agarose microchamber array chip to evaluate the effect of haloperidol on mouse CM beating activity (166).The authors developed two systems, a four-cell and a nine-cell CM network.They observed that the beating rhythm of the CMs were unstable and slower after the addition and wash out of haloperidol in the four-CM network because this system was not large enough to re-establish a stable beating state.Instead, the beating rhythm was returned to its original state after the haloperidol wash in the nine-cell network.These results showed the importance of tissue culture size on the stability of drug screening models.Song et al.
reported a diabetic cardiac tissue to test the effect of antidiabetic thiazolidinedione drugs.
For that purpose, the authors cultured rat CMs in collagen gels under different media conditions to reproduce a diabetic myocardium (167).The authors studied the CMs under normal, diabetic, and therapeutic conditions.Their results showed that CMs cultivated with high glucose media showed low current propagation and high cell apoptosis.
Thiazolidinedione enhanced electrical properties and viability of CMs.The proposed platform is suitable for drug screening on engineered cardiac tissues in diabetic conditions.
Functional CMs can be obtained from human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) (168)(169)(170)(171)(172)(173).Despite their immature characteristics, extensive pharmacological studies have The device was fabricated using the conventional lithographic technique and molded in PDMS.They then exposed the human CMs to hydrogen peroxide (H2O2) to mimic the oxidative stress implicated in various disease states and observed that contractile activity was dramatically suppressed.
Many recent studies focused on engineering cardiac tissue constructs from iPSC-derived CMs (178)(179)(180).Cardiac muscle tissues obtained from human iPSCs combined with microfluidic systems has become a useful platform to improve preclinical pharmacology as well as drug discovery approaches.Incorporating vasculature in engineered cardiac tissue is needed to enhance physiological fidelity and enhance tissue survival.George and colleges created perfused human capillary networks from human endothelial colony forming cell-derived ECs (Figure 5) (181).Later, the authors combined the network of human capillaries in the presence of cardiac muscle spheroids derived from human iPSCs to fabricate a vascularized cardiac microtissue (Figure 5-C) (182).Their results showed that human CMs survived and continued to contract within the device for up to 28 days, while a surrounding vessel network was developed.
Mathur et al. developed a human cardiac microphysiological system from human iPSCderived CMs for pharmacological studies (183).The novel device contained a cell chamber, two neighboring channels for medium, and some connecting microchannels mimicking the endothelial barrier.This barrier protected the muscle tissue from shear forces and allowed the diffusion of nutrients.The authors obtained robust and reproducible human 3D aligned cardiac microtissues modifying a previous study in serum-free media (184).Human iPSC-derived CMs started beating spontaneously and without any external stimulation.After 7 days, multiple cell layers started to contract in a uniaxial manner.The engineered cardiac tissue was tested with four model drugs (verapamil, E-4031, isoproterenol, and metoprolol).The results showed a good correlation of tissue response to those previously recorded with other methods in human cardiac cells.For example, the addition of isoproterenol enhanced the tissue beating.All aforementioned investigations show a great progress in the development of reliable cardiac tissues in vitro for disease modeling and drug screening applications.Moreover, they emphasize that the cardiac tissue-on-chip models generally have better prediction of clinical results compared to conventional 2D culture models.Our lab recently developed a tissue construct named "AngioChip", the most advanced vascularized cardiac platform to date, which combined multiple layers of branched networks (Figure 2-E).Microholes and nanopores within the tissue scaffold were prepared to fortify permeability and cell migration.Developed cardiac tissues with AngioChip technology were implanted in vivo and successfully connected with the vasculature system of the host (185).

Skeletal muscle
In general, skeletal muscle tissue engineering uses cells, scaffolds, and growth factors aiming to regenerate native skeletal muscles (186).Several cell types, such as satellite cells, myoblasts, mesoangioblasts, pericytes, embryonic stem cells, mesenchymal stem cells, and iPSCs have been identified as potential sources for cells for skeletal muscle regeneration (117,187).In addition, different methods to construct 3D skeletal muscles in vitro have been developed with the ability to apply both mechanical and electrical stimulations (188).These techniques focus on scaffold-free concepts (189)(190)(191) or on scaffold-based approaches (192)(193)(194)(195). Numerous groups have documented the use of 3D skeletal muscle tissues as in vitro models for pharmacological screening tests or biological studies (196)(197)(198).For example, Syverud et al. developed 3D skeletal muscle units (SMUs) and examined the potential of dexamethasone as a growth factor (199).
SMUs were tissue constructs fabricated from contractile myotubes and monolayers of primary fibroblasts.The results showed that dexamethasone improved myogenic proliferation and myotube fusion when it was added before induction of differentiation.
Moreover, SMUs exposed to 10 nM of dexamethasone on day 6 or day 0 showed organized muscle structure and enhanced force production.
Tourovskaia et al. fabricated a microfluidic platform to stimulate myotubes with agrin, a chemical expressed by neurons in vivo at the nerve-muscle junction (200,201).The microfluidic system was fabricated using soft lithography in PDMS and consisted of a main channel formed by different numbers of converging inlet channels for the delivery of drugs to the myotubes.The results suggested that agrin is an important stabilizer in the synapse formation.This microfluidic device allowed an accurate control of perfusion and chemical environment surrounding cells.In addition, the device can be used for studies of spatiotemporal competition between different stimuli.In another work, Anene-Nzelu et al. developed 3D microfluidic systems with microtopographical cues to obtain aligned myoblast constructs (202).The top layer of the system consisted of a microfluidic channel with a micropillar array to immobilize C2C12 cells and a bottom PDMS microgrooved layer.The authors observed that C2C12 cells aligned along the direction of microgrooves.
In addition, it was observed that the alignment enhanced the maturation of myoblast constructs and the expression of skeletal muscle genes.The proposed microfluidic system is simple, cost-effective, and scalable and can be used as a powerful platform for drug screening.
Although several strategies have been proposed to make contractile 3D skeletal muscles in vitro, Vandenburgh et al. made the most significant contribution toward using engineered muscle tissues in drug screening.They developed miniature bioartificial muscles (mBAMs) in 96-well plates (203,204).mBAMs were 3D contractile tissues with organized and striated skeletal muscle fibers, which can generate directed force when electrically stimulated.In their model, murine myoblasts were mixed with collagen, Matrigel, or fibrinogen-thrombin gels and cast in a flexible well with microposts on each side (Figure 6).This approach provided a nondestructive and sensitive method for measuring muscle contractile forces.Preliminary work tested Atorvastatin and insulinlike growth factor-1 on healthy murine muscle fibers (205).The results demonstrated mBAM myofiber active force and hypertrophy were enhanced as the muscle tissues were exposed to insulin-like growth factor-1.However, mBAM weakness and deterioration were reported for the tissues exposed to a cholesterol-lowering statin.Following this work, the system was automated and was used for high-throughput screening experiments (206).
The author tested the effects of 31 potential dugs on mdx mouse-derived mBAMs to treat Duchenne muscular dystrophy.The results showed that 11 compounds enhanced the muscle force generation.The novel automated system can be used as a powerful preclinical tool in pharmaceutical applications.
Recently, Madden et al. fabricated human skeletal muscle culture systems, named myobundles, using primary myogenic cells to study their contractile activity and biochemical responses to three different drugs (207).Myogenic cells were derived from human muscle biopsy and human myobundles were generated using a hydrogel molding method previously described with rat cells (208).After 5 days of differentiation, the myofibers showed spontaneous contraction.Human myobundles were tested with statins (cerivastatin and lovastatin), chloroquine, and clenbuterol and their contractile and biochemical responses were recorded.The results showed that myobundles exhibited aligned architecture, multinucleated, and striated myofibers.Also, they contracted spontaneously and responded to electrical and biochemical stimuli.The authors observed that myobundles were more sensitive to cerivastatin than lovastatin, which agrees with previous clinical reports.In response to chloroquine, myobundles induced the autophagic myopathy observed in muscle tissues in vivo.Clenbuterol at low concentration caused the myofiber hypertrophy and increased contractile strength of myobundles.However, higher concentrations of clenbuterol led to muscle weakness consistent with previous animal and human studies.In general, these studies suggested that myobundles were able to recapitulate the physiological responses of native muscles and can be used as a preclinical tool for predictive pharmacological screening.

Conclusion
This review briefly described the morphology and structure of skeletal and cardiac muscle tissues.Commonly used approaches in fabricating muscle tissues were explained providing a similar natural ECM for muscle cells in vitro.For example, aligned electrospun sheets guided cellular alignment that is necessary for muscle functionality.
Bioprinting methods are able to provide rapid fabrication, cellular viability in 3D PDMS elastomers (Figure4-A).They assembled eight separate MTFs on a single device.With this novel system, the authors were able to quantify the contractility of several cardiac muscle tissues simultaneously (Figure4-B).Two years later, Agarwal et al. modified the heart-on-a-chip system and used it to test isoproterenol on cardiac muscle contractility(160).The authors developed semiautomatic device incorporating a system to control drug injection, temperature, and electrical stimulation (Figure4-C).The device consisted of a simple microfluidic channel, which is reusable, transparent, and biocompatible and did not require specialized skills to use it.The microdevice was tested with continuous perfusion of isoproterenol at different exposures during electrical stimulation of MTF.This new device is suitable for high-content drug testing in cardiac muscle tissues in vitro.Eschenhagen and Zimmerman developed a cardiac model, called engineered heart tissue (EHT), employing embryonic chick CMs suspended in a collagen matrix(162).Some years after, the same research group reported improvements of their device and used neonatal rat CMs in a Matrigel/collagen gel(163).The authors prepared 3D circular EHTs by culturing CMs around a central Teflon cylinder.Compared to other techniques for engineering cardiac muscles, this system exhibited a higher cardiac tissue/matrix ratio, contractility, and maturation.Hansen et al. modified their device to obtain mini-EHTs in a fibrinogen/thrombin/Matrigel matrix(145).The authors used fibrinogen and thrombin instead of collagen because the polymerization of hydrogel was more rapid than the gelation of collagen.The authors used the ring format instead of a strip format in creating mini-EHTs.Posteriorly, the 24-well mini-EHTs format was used to evaluate 46 proarrhythmic drugs under perfusion and/or electrically stimulation on the contractile activity of muscle tissues(164).The study was carried out with 14 to 21-day old mini-EHTs and three different concentrations of drugs.The authors observed that the mini-EHTs were sensitive to the drug concentrations.Recently, Eschenhagen group utilized the EHTs to explore tyrosine kinase inhibitors-mediated cardiotoxicity using immunohistology and transmission electron microscopy(165).
constructs, and the ability to examine muscle cell behaviors and function, such as elongation and proliferation.DEP was another microscale technology for alignment and accurate positioning of cells.Finally, textile technology and microfluidics with the ability to produce commercialized cardiac support devices and grafts revealed the clinical advantage of tissue engineering.Following this, we discussed commonly developed cardiac and muscle tissues for drug screening purposes.The advent of tissue-on-a-chip platforms is a promising approach to mimic the in vivo-like environment for muscle tissues in a miniaturized, low cost, and scalable manner.Muscle cell differentiation, alignment, contractile activity, and tissue formation are characteristics that most of the experiments determine as a function of tissue maturation and performance.Despite great work on tissue fabrication and testing for drug discovery, further research and innovation are required prior to widely used and successful commercialization of engineered tissues in pharmaceutical industry.214.Liu J, Gao C, Chen W, Ma W, Li X, Shi Y, et al.CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation.Transl.Psychiatry 2016; 6(1): e703.215.Obregón R, Ahadian S, Ramón-Azcón J, Chen L, Fujita T, Shiku H, et al.Noninvasive measurement of glucose uptake of skeletal muscle tissue models using a glucose nanobiosensor.Biosens.Bioelectron.2013; 50: 194-201.216.Pelfrey S, Cantu T, Papantonakis MR, Simonson DL, McGill RA, Macossay J. Microscopic and spectroscopic studies of thermally enhanced electrospun PMMA microand nanofibers.Polym Chem.2010; 1(6): 866-9.

Figure 1 .
Figure 1.Anatomy of cardiac and skeletal muscle in the body.(A) Cardiac muscle.(B) Skeletal muscle.

Figure 2 .
Figure 2. Methods to fabricate muscle tissues in vitro.(A) The schematic of electrospinning setup (216).(B) Integrated tissue-organ printer system as an example of bioprinting approach(54).(C) The creation process inside GelMA hydrogel for CNT alignment using DEP(71).(D) Fabrication of meter-long cell-laden microfiber process using textile technology(78).(E) A schematic of AngioChip platform made with microfluidic technique(185).

Figure 3 .
Figure 3.In vitro parameters for mimicking in vivo environment.(A) Before and after applying electrical stimulation to C2C12 myotubes.(B) The mean percent of aligned C2C12 myotubes for different regimes (voltage for regime 1 and 2 were 0.5 and 6 respectively) (117).(C) Aligning C2C12 cells by applying mechanical and electrical cues (71).

Figure 4 .
Figure 4. Heart-on-a-chip models.(A) Fabrication procedure of contractile CM stripes.(B) Time-lapse bright-field pictures of contractile CM stripes.Analysis of CM contractility (PDMS layer=18.6µm): (i) Bright-field images of films adhered to the substrate, (ii) films curved at diastole and peak systole, and (iii) the film length (blue) and x-projection (red) placed on the tissue pictures.Scale bar: 5 mm.(C) Schematics of the fabrication procedure for MTF and semiautomatic microdevice integrated a MTF chip.
Recent advances in biomaterial synthesis, stem cell biology, and microfabrication technologies have enabled researchers to develop biomimetic and physiologically relevant muscle tissue constructs for drug screening and disease modeling applications.electrodes (215) can provide powerful tools in characterization of highthroughput tissue models in an accurate and non-destructive manner.
(211)ntrations on a single tissue, simultaneously.Therefore, time and cost associated to tissue-based drug screening platforms can substantially be decreased.Moreover, novel analytical methods or materials, such as scanning electrochemical microscopy(211)and nanoporous gold