Skeletal Muscle Tissue Engineering Research Articles

Porous biomaterial scaffolds for skeletal muscle tissue engineering.

Volumetric muscle loss is a traumatic injury which overwhelms the innate repair mechanisms of skeletal muscle and results in significant loss of muscle functionality. Tissue engineering seeks to regenerate these injuries through implantation of biomaterial scaffolds to encourage endogenous tissue formation and to restore mechanical function. Many types of scaffolds are currently being researched for this purpose. Scaffolds are typically made from either natural, synthetic, or conductive polymers, or any combination therein. A major criterion for the use of scaffolds for skeletal muscle is their porosity, which is essential for myoblast infiltration and myofiber ingrowth. In this review, we summarize the various methods of fabricating porous biomaterial scaffolds for skeletal muscle regeneration, as well as the various types of materials used to make these scaffolds. We provide guidelines for the fabrication of scaffolds based on functional requirements of skeletal muscle tissue, and discuss the general state of the field for skeletal muscle tissue engineering.

Real‐time and Multichannel Measurement of Contractility of hiPSC‐Derived 3D Skeletal Muscle using Fiber Optics‐Based Sensing

AbstractAs the field of cardiac and skeletal muscle tissue engineering expands, so does the need for accurate and reliable systems to generate in vitro 3D tissues and analyze their functional properties. In this study, the Cuore is introduced, a system that integrates sensors based on optical fibers and uses the principle of light interferometry to detect the contraction of 3D Tissue Engineered Skeletal Muscles (3D‐TESMs). The technology employed in the Cuore allows for reproducible and multichannel force measurements down to a nano‐Newtons resolution while maintaining sterility and permitting continuous non‐invasive recording within and outside standard tissue culture incubators. Thanks to the integrated electrodes for electrical pulse stimulation (EPS), 3D‐TESMs generated from three independent hiPSC‐derived myogenic progenitors (MPs) lines are stimulated and the contractility is recorded over the course of a week. Through the modulation of different EPS parameters, the optimal combination to induce the 3D‐TESMs in producing fully fused tetani without causing damage is determined. Furthermore, 3D‐TESMs from different lines exhibit characteristic signatures of spontaneous contractility and response to caffeine, verapamil, and the β‐agonist clenbuterol. The ease of use, high sensitivity, and the integrated electrodes and sensors make the Cuore an ideal technology to investigate the biology of contractile tissues and their response to drugs.

Tunable Light-Actuated Interpenetrating Networks of Silk Fibroin and Gelatin for Tissue Engineering and Flexible Biodevices.

Soft materials with tunable properties are valuable for applications such as tissue engineering, electronic skins, and human-machine interfaces. Materials that are nature-derived offer additional advantages such as biocompatibility, biodegradability, low-cost sourcing, and sustainability. However, these materials often have contrasting properties that limit their use. For example, silk fibroin (SF) has high mechanical strength but lacks processability and cell-adhesive domains. Gelatin, derived from collagen, has excellent biological properties, but is fragile and lacks stability. To overcome these limitations, composites of gelatin and SF have been explored. However, mechanically robust self-supported matrices and electrochemically active or micropatterned substrates were not demonstrated. In this study, we present a composite of photopolymerizable SF and photogelatin, termed photofibrogel (PFG). By incorporating photoreactive properties in both SF and gelatin, control over material properties can be achieved. The PFG composite can be easily and rapidly formed into free-standing, high-resolution architectures with tunable properties. By optimizing the ratio of SF to gelatin, properties such as swelling, mechanical behavior, enzymatic degradation, and patternability are tailored. The PFG composite allows for macroscale and microscale patterning without significant swelling, enabling the fabrication of structures using photolithography and laser cutting techniques. PFG can be patterned with electrically conductive materials, making it suitable for cell guidance and stimulation. The versatility, mechanical robustness, bioactivity, and electrochemical properties of PFG are shown for skeletal muscle tissue engineering using C2C12 cells as a model. Overall, such composite biomaterials with tunable properties have broad potential in flexible bioelectronics, wound healing, regenerative medicine, and food systems.

Cryopreservation Alters Tissue Structure and Improves Differentiation of Engineered Skeletal Muscle.

Tissue-engineered skeletal muscle can play an important role in regenerative medicine, disease modeling, drug testing, as well as the actuation of biohybrid machines. As the applications of engineered muscle tissues expand, there exists a growing need to cryopreserve and store these tissues without impairing function. In a previous study, we developed a cryopreservation protocol in which engineered skeletal muscle tissues are frozen before myogenic differentiation. In that study, we found that this cryopreservation process led to a three-fold increase in the force generation of the differentiated muscle. Here, we perform further testing to determine the mechanisms by which cryopreservation enhances engineered skeletal muscle function. We found that cryopreservation alters the microstructure of the tissue by increasing pore size and decreasing elastic modulus of the extracellular matrix (ECM), which leads to increased expression of genes related to cell migration, cell-matrix adhesion, ECM secretion, and protease activity. Specifically, cryopreservation leads to the upregulation of many ECM proteins, including laminin, fibronectin, and several types of collagens, as well as integrins and matrix metalloproteinases. These changes to ECM structure and composition were associated with enhanced myogenic differentiation, as evidenced by the upregulation of late-stage myogenic markers and increased force generation. These results highlight the need to understand the effects of cryopreservation on the ECM of other tissues as we strive to advance tissue and organ cryopreservation protocols for regenerative medicine.

Diabetes diminishes muscle precursor cell-mediated microvascular angiogenesis.

The skeletal muscles of Type II diabetic (T2D) patients can be characterized by a reduced vessel density, corresponding to deficiencies in microvascular angiogenesis. Interestingly, T2D also inhibits the function of many myogenic cells resident within skeletal muscle, including satellite cells, which are well-known for the role they play in maintaining homeostasis. The current study was undertaken to gain a better understanding of the mechanisms whereby satellite cell progeny, muscle precursor cells (MPCs), influence microvascular angiogenesis. Network growth and the expression of genes associated with angiogenesis were reduced when microvessels were treated with conditioned media generated by proliferating MPCs isolated from diabetic, as compared to control rat skeletal muscle, a phenomenon that was also observed when myoblasts from control or diabetic human skeletal muscle were used. When only exosomes derived from diabetic or control MPCs were used to treat microvessels, no differences in microvascular growth were observed. An evaluation of the angiogenesis factors in control and diabetic MPCs revealed differences in Leptin, vascular endothelial growth factor (VEGF), IL1-β, interleukin 10, and IP-10, and an evaluation of the MPC secretome revealed differences in interleukin 6, MCP-1, VEGF, and interleukin 4 exist. Angiogenesis was also reduced in tissue-engineered skeletal muscles (TE-SkM) containing microvessels when they were generated from MPCs isolated from diabetic as compared to control skeletal muscle. Lastly, the secretome of injured control, but not diabetic, TE-SkM was able to increase VEGF and increase microvascular angiogenesis. This comprehensive analysis of the interaction between MPCs and microvessels in the context of diabetes points to an area for alleviating the deleterious effects of diabetes on skeletal muscle.

Long-term longitudinal study on swine VML model

BackgroundVolumetric Muscle Loss (VML), resulting from severe trauma or surgical ablation, is a pathological condition preventing myofibers regeneration, since skeletal muscle owns the remarkable ability to restore tissue damage, but only when limited in size. The current surgical therapies employed in the treatment of this pathology, which particularly affects military personnel, do not yet provide satisfactory results. For this reason, more innovative approaches must be sought, specifically skeletal muscle tissue engineering seems to highlight promising results obtained from preclinical studies in VML mouse model. Despite the great results obtained in rodents, translation into human needs a comparable animal model in terms of size, in order to validate the efficacy of the tissue engineering approach reconstructing larger muscle mass (human-like). In this work we aim to demonstrate the validity of a porcine model, that has underwent a surgical ablation of a large muscle area, as a VML damage model.ResultsFor this purpose, morphological, ultrasound, histological and fluorescence analyses were carried out on the scar tissue formed following the surgical ablation of the peroneus tertius muscle of Sus scrofa domesticus commonly called mini-pig. In particular, the replenishment of the damaged area, the macrophage infiltration and the vascularization at different time-points were evaluated up to the harvesting of the scar upon six months.ConclusionHere we demonstrated that following VML damage, there is an extremely poor regenerative process in the swine muscle tissue, while the formation of fibrotic, scar tissue occurs. The analyses performed up to 180 days after the injury revealed the development of a stable, structured and cellularized tissue, provided with vessels and extracellular matrix acquiring the status of granulation tissue like in human.

Effect of 3D‐Printable Anisotropic Fibrous Hydrogels on Fabricating Artificial Skeletal Muscle Constructs

AbstractThe use of fibrous hydrogel constructs is a promising strategy in tissue engineering because of their ability to closely mimic the anisotropic structure of biological tissues. In skeletal muscle tissue engineering, fiber alignment is considered a crucial factor for achieving muscle functions originating from the uniaxially aligned structure of myofibers; however, the fabrication process of hydrogel constructs with aligned fibers requires complex and laborious techniques. In this study, the effect of 3D‐printed constructs with randomly oriented fibers on myoblast differentiation is evaluated. To this end, 3D‐printable gelatin methacryloyl (GelMA) hydrogel microfibers through the mechanical fragmentation of electrospun GelMA sheets is prepared. The bulk rheological and tensile properties of the hydrogels with microfibers are stronger than those of the hydrogels without microfibers. Interestingly, when myoblast‐embedded constructs are 3D‐printed with and without randomly oriented GelMA hydrogel microfibers and incubated for cell differentiation, the microfibers in the constructs effectively promote myotubule‐like cell formation. In addition, the uniaxial 3D printed design enables anisotropic myotubule formation. This shows that hydrogels with only fibers distributed randomly can be applied to achieve more effective artificial muscle constructs than those achieved using normal bulk hydrogels and are easier to use than aligned fibers.

3D Co-Printing and Substrate Geometry Influence the Differentiation of C2C12 Skeletal Myoblasts.

Cells are influenced by several biomechanical aspects of their microenvironment, such as substrate geometry. According to the literature, substrate geometry influences the behavior of muscle cells; in particular, the curvature feature improves cell proliferation. However, the effect of substrate geometry on the myogenic differentiation process is not clear and needs to be further investigated. Here, we show that the 3D co-printing technique allows the realization of substrates. To test the influence of the co-printing technique on cellular behavior, we realized linear polycaprolactone substrates with channels in which a fibrinogen-based hydrogel loaded with C2C12 cells was deposited. Cell viability and differentiation were investigated up to 21 days in culture. The results suggest that this technology significantly improves the differentiation at 14 days. Therefore, we investigate the substrate geometry influence by comparing three different co-printed geometries-linear, circular, and hybrid structures (linear and circular features combined). Based on our results, all structures exhibit optimal cell viability (>94%), but the linear pattern allows to increase the in vitro cell differentiation, in particular after 14 days of culture. This study proposes an endorsed approach for creating artificial muscles for future skeletal muscle tissue engineering applications.

Induced Pluripotent Stem Cells for Tissue-Engineered Skeletal Muscles.

Skeletal muscle, which comprises a significant portion of the body, is responsible for vital functions such as movement, metabolism, and overall health. However, severe injuries often result in volumetric muscle loss (VML) and compromise the regenerative capacity of the muscle. Tissue-engineered muscles offer a potential solution to address lost or damaged muscle tissue, thereby restoring muscle function and improving patients' quality of life. Induced pluripotent stem cells (iPSCs) have emerged as a valuable cell source for muscle tissue engineering due to their pluripotency and self-renewal capacity, enabling the construction of tissue-engineered artificial skeletal muscles with applications in transplantation, disease modelling, and bio-hybrid robots. Next-generation iPSC-based models have the potential to revolutionize drug discovery by offering personalized muscle cells for testing, reducing reliance on animal models. This review provides a comprehensive overview of iPSCs in tissue-engineered artificial skeletal muscles, highlighting the advancements, applications, advantages, and challenges for clinical translation. We also discussed overcoming limitations and considerations in differentiation protocols, characterization methods, large-scale production, and translational regulations. By tackling these challenges, iPSCs can unlock transformative advancements in muscle tissue engineering and therapeutic interventions for the future.

Fabrication of electrospun nanofibrous clinoptilolite doped thermoplastic polyurethane scaffolds for skeletal muscle tissue engineering

AbstractThe treatment of skeletal muscle, which lost its function with damage or trauma with autologous muscle tissue transfer, is a very problematic approach. Hence, it is critical to develop materials that are mimicking muscle tissue mechanical behaviors and allowing cell adhesion. Polyurethanes (PUs) are one of the most common polymers in tissue engineering applications and skeletal muscle regeneration due to their elasticity and mechanical flexibility. Clinoptilolite (CLN) is a hydrated alumina silica crystal based biocompatible material that numerous positive effects on animal and human health. Here, we report the synthesize of flexible membranes based on clinoptilolite (CLN) doped thermoplastıc polyurethane (TPU) nanofiber network to be used in the field of skeletal muscle regeneration. We primarily evaluated their ability to mimic skeletal muscle by determining their mechanical properties and cell adhesion rates. We observe that cell adhesion and proliferation increased with the increase of CLN contribution. Young modulus (EY) values of pure TPU, 5 and 10 wt.% CLN‐doped TPU fibers are 3.66, 2.37, and 1.85 MPa, respectively. Mechanical elongations at break of pure TPU, 5 and 10 wt.% CLN‐doped TPU fibers after 37°C treatment (7th day) are 193.41%, 113.30%, and 197.15%, respectively. With the addition of 5 wt.% CLN, the thermal stability slightly increased compared to the pure TPU and 10 wt.% CLN/TPU. In addition, cytotoxicity studies reveal that CLN/PU membranes are biocompatible, and finally cell adhesion increases proportionally to the increased CLN contribution. The obtained results indicate that the CLN/PU membranes can be used as a skeletal muscle scaffold.

Myogenic differentiation of human myoblasts and Mesenchymal stromal cells under GDF11 on Poly-ɛ-caprolactone-collagen I-Polyethylene-nanofibers

BackgroundFor the purpose of skeletal muscle engineering, primary myoblasts (Mb) and adipogenic mesenchymal stem cells (ADSC) can be co-cultured and myogenically differentiated. Electrospun composite nanofiber scaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining both biocompatibility and stability Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoring skeletal muscle function in aging mice, some studies have also described a harming effect of GDF11. Therefore, the aim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolactone (PCL)-collagen I-polyethylene oxide (PEO)-nanofibers.ResultsHuman Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-PEO-nanofibers. Differentiation media were either serum-free with or without GDF11, or serum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity. Immunofluorescence staining showed myosine heavy chain expression in all groups after 28 days of differentiation without any clear evidence of more or less pronounced expression in either group. Gene expression of myosine heavy chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone.ConclusionsThis is the first study analyzing the effect of GDF11 on myogenic differentiation of Mb and ADSC co-cultures under serum-free conditions. The results of this study show that PCL-collagen I-PEO-nanofibers represent a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidence of a harming effect.

Modulation of myoblast differentiation by electroactive scaffold morphology and biochemical stimuli

The physico-chemical properties of the scaffold materials used for tissue regeneration strategies have a direct impact on cell shape, adhesion, proliferation, phenotypic and differentiation. Herewith, biophysical and biochemical cues have been widely used to design and develop biomaterial systems for specific tissue engineering strategies. In this context, the patterning of piezoelectric polymers that can provide electroactive stimuli represents a suitable strategy for skeletal muscle tissue engineering applications once it has been demonstrated that mechanoelectrical stimuli promote C2C12 myoblast differentiation. In this sense, this works reports on how C2C12 myoblast cells detect and react to physical and biochemical stimuli based on micropatterned poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) electroactive scaffolds produced by soft lithography in the form of arrays of lines and hexagons (anisotropic and isotropic morphology, respectively) combined with differentiation medium. The scaffolds were evaluated for the proliferation and differentiation of C2C12 myoblast cell line and it is demonstrated that anisotropic microstructures promote muscle differentiation which is further reinforced with the introduction of biochemical stimulus. However, when the physical stimulus is not adequate to the tissue, e.g. isotropic microstructure, the biochemical stimulus has the opposite effect, hindering the differentiation process. Therefore, the proper morphological design of the scaffold combined with biochemical stimulus allows to enhance skeletal muscle differentiation and allows the development of advanced strategies for effective muscle tissue engineering.

Retracted: A Photoclick Thiol‐Ene Collagen‐Based Hydrogel Platform for Skeletal Muscle Tissue Engineering

AbstractUV‐cured collagen‐based hydrogels hold promise in skeletal muscle regeneration due to their soft elastic properties and porous architecture. However, the complex triple helix conformation of collagen and environmental conditions, i.e., molecular oxygen, pose risks to reaction controllability, wet‐state integrity, and reproducibility. To address this challenge, a photoclick hydrogel platform is presented through an oxygen‐insensitive thiol‐ene reaction between 2‐iminothiolane (2IT)‐functionalized type I collagen and multiarm, nonhomopolymerizable norbornene‐terminated polyethylene glycol (PEG). UV‐induced network formation is demonstrated by oscillatory time sweeps on the reacting thiol‐ene mixture, so that significantly increased storage moduli are measured and adjusted depending on the photoinitiator concentration. Variations in PEG functionality (4‐arm and 8‐arm) and PEG content generate hydrogels with skeletal muscle native stiffness (Ec = 1.3 ± 0.2‒11.5 ± 0.9 kPa), diffusion‐controlled swelling behavior and erosion‐driven degradability. In vitro, no cytotoxic effect is detected on C2C12 murine myoblasts, while myogenic differentiation is successfully accomplished on hydrogel‐seeded cells in then low serum culture medium. In vivo, 7 d subcutaneous implantation of selected thiol‐ene hydrogel in rats reveal higher cell infiltration, blood vessel formation, and denser tissue interface compared to a clinical gold standard collagen matrix (Mucograft, a trademark of Geistlich Biomaterials). These results, therefore, support the applicability and further development of this hydrogel platform for skeletal muscle regeneration.

A physically cross-linked double network polysaccharides/Ca2+ hydrogel scaffold for skeletal muscle tissue engineering

The current therapy for muscle injury is facing challenges in tissue self-regeneration and function restoration, which declines steeply with age. Tissue engineering is a promising strategy for treating skeletal muscular disorders. Natural polysaccharides have great potential to support cell growth and attachment as tissue-engineered scaffolds. A three-dimensional (3D) porous hydrogel scaffold has been prepared based on the double network of natural polysaccharide sodium alginate (SA), chitosan (CS) and Ca2+ ions through the freeze-drying process. The physical, mechanical, and biological properties of the CS/SA/Ca2+ hydrogel scaffold can be controlled by adjusting the SA/CS ratio. The physically cross-linked double network hydrogel showed good biosafety, high stability and satisfactory degradation rate. When the CS/SA molar ratio was 1:1.2 (H-1.2 group), the pore size and compression modulus of the double network hydrogel were 104.06 ± 2.64 µm and 40.00 ± 2.52 kPa, respectively. The results of in vitro evaluation showed that the 3D structure of the CS/SA/Ca2+ double network hydrogel could well support adhesion and proliferation of C2C12 murine myoblasts and porcine skeletal muscle stem cells. The best cell growth effect was achieved in the H-1.2 group. The physically cross-linked CS/SA/Ca2+ double network hydrogel can be a promising printable material as the cell scaffold in applications of skeletal muscle tissue engineering.

Electrically Triggered Quercetin Release from Polycaprolactone/Bismuth Ferrite Microfibrous Scaffold for Skeletal Muscle Tissue.

Skeletal muscle tissue engineering presents a promising avenue to address the limitations pertaining to the regenerative potential of stem cells in case of injury or damage. The objective of this research was to evaluate the effects of utilizing novel microfibrous scaffolds, containing the compound quercetin (Q), on skeletal muscle regeneration. Morphological test results showed us that the combination of bismuth ferrite (BFO), polycaprolactone (PCL), and Q were bonded and well-ordered with each other, and a uniform microfibrous structure was obtained. Antimicrobial susceptibility testing of PCL/BFO/Q was conducted, and microbial reduction was found to be over 90% in the highest concentration of Q-loaded microfibrous scaffolds with the most inhibitory effect on S. aureus strains. Further, biocompatibility was investigated by performing MTT testing, fluorescence testing, and SEM imaging on mesenchymal stem cells (MSCs) to determine whether they could act as suitable microfibrous scaffolds for skeletal muscle tissue engineering. Incremental changes in the concentration of Q led to increased strength and strain, allowing muscles to withstand stretching during the healing process. In addition, electrically conductive microfibrous scaffolds enhanced the drug release capability by revealing that Q can be released significantly more quickly by applying the appropriate electric field, compared with conventional drug-release techniques. These findings suggest a possible use for PCL/BFO/Q microfibrous scaffolds in skeletal muscle regeneration by demonstrating that the combined action of both guidance biomaterials was more successful than Q itself acting alone.

Cyclic Tensile Stress Induces Skeletal Muscle Hypertrophy and Myonuclear Accretion in a 3D Model.

Skeletal muscle is highly adaptive to mechanical stress due to its resident stem cells and the pronounced level of myotube plasticity. Herein, we study the adaptation to mechanical stress and its underlying molecular mechanisms in a tissue-engineered skeletal muscle model. We subjected differentiated 3D skeletal muscle-like constructs to cyclic tensile stress using a custom-made bioreactor system, which resulted in immediate activation of stress-related signal transducers (Erk1/2, p38). Cell cycle re-entry, increased proliferation, and onset of myogenesis indicated subsequent myoblast activation. Furthermore, elevated focal adhesion kinase and β-catenin activity in mechanically stressed constructs suggested increased cell adhesion and migration. After 3 days of mechanical stress, gene expression of the fusogenic markers MyoMaker and MyoMixer, myotube diameter, myonuclear accretion, as well as S6 activation, were significantly increased. Our results highlight that we established a promising tool to study sustained adaptation to mechanical stress in healthy, hypertrophic, or regenerating skeletal muscle. Impact statement Sustained adaption to mechanical stress presents a key feature for skeletal muscle functionality and growth. Knowledge of these processes, however, is mostly based on in vivo or 2D cell culture models, both of which entail significant shortcomings. Herein, we generated highly hypertrophic tissue-engineered skeletal muscle-like constructs that are comparable to the results of successful in vivo models of adaption to mechanical stimuli, achieving an outcome that only few in vitro approaches have reached. Second, we aimed at studying the underlying molecular mechanisms, which is of interest since there is little knowledge of the intracellular events during hypertrophy upon mechanical stimulation.

The Use of Collagen Methacrylate in Actuating Polyethylene Glycol Diacrylate-Acrylic Acid Scaffolds for Muscle Regeneration.

After muscle loss or injury, skeletal muscle tissue has the ability to regenerate and return its function. However, large volume defects in skeletal muscle tissue pose a challenge to regenerate due to the absence of regenerative elements such as biophysical and biochemical cues, making the development of new treatments necessary. One potential solution is to utilize electroactive polymers that can change size or shape in response to an external electric field. Poly(ethylene glycol) diacrylate (PEGDA) is one such polymer, which holds great potential as a scaffold for muscle tissue regeneration due to its mechanical properties. In addition, the versatile chemistry of this polymer allows for the conjugation of new functional groups to enhance its electroactive properties and biocompatibility. Herein, we have developed an electroactive copolymer of PEGDA and acrylic acid (AA) in combination with collagen methacrylate (CMA) to promote cell adhesion and proliferation. The electroactive properties of the CMA + PEGDA:AA constructs were investigated through actuation studies. Furthermore, the biological properties of the hydrogel were investigated in a 14-day in vitro study to evaluate myosin light chain (MLC) expression and metabolic activity of C2C12 mouse myoblast cells. The addition of CMA improved some aspects of material bioactivity, such as MLC expression in C2C12 mouse myoblast cells. However, the incorporation of CMA in the PEGDA:AA hydrogels reduced the sample movement when placed under an electric field, possibly due to steric hindrance from the CMA. Further research is needed to optimize the use of CMA in combination with PEGDA:AA as a potential scaffold for skeletal muscle tissue engineering.

A novel extrusion-based 3D bioprinting system for skeletal muscle tissue engineering

Three-dimensional (3D) bioprinting is an emerging technology, which turned out to be an optimal tool for tissue engineering approaches. To date, different printing systems have been developed. Among them, the extrusion-based approach demonstrated to be the most suitable for skeletal muscle tissue engineering, due to its ability to produce and deposit printing fibers in a parallel pattern that well mimic the native skeletal muscle tissue architecture. In tissue bioengineering, a key role is played by biomaterials, which must possess the key requisite of ‘printability’. Nevertheless, this feature is not often well correlated with cell requirements, such as motives for cellular adhesion and/or absorbability. To overcome this hurdle, several efforts have been made to obtain an effective bioink by combining two different biomaterials in order to reach a good printability besides a suitable biological activity. However, despite being efficient, this strategy reveals several outcomes limitations. We report here the development and characterization of a novel extrusion-based 3D bioprinting system, and its application for correction of volumetric muscle loss (VML) injury in a mouse model. The developed bioprinting system is based on the use of PEG-Fibrinogen, a unique biomaterial with excellent biocompatibility, well-suited for skeletal muscle tissue engineering. With this approach, we obtained highly organized 3D constructs, in which murine muscle progenitors were able to differentiate into muscle fibers arranged in aligned bundles and capable of spontaneously contracting when cultured in vitro. Furthermore, to evaluate the potential of the developed system in future regenerative medicine applications, bioprinted constructs laden with either murine or human muscle progenitors were transplanted to regenerate the Tibialis Anterior muscle of a VML murine model, one month after grafting.

Bioactive nanoglass regulating the myogenic differentiation and skeletal muscle regeneration.

Bioactive glass nanoparticles (BGNs) are widely used in the field of biomedicine, including drug delivery, gene therapy, tumor therapy, bioimaging, molecular markers and tissue engineering. Researchers are interested in using BGNs in bone, heart and skin regeneration. However, there is inadequate information on skeletal muscle tissue engineering, limited information on the biological effects of BGNs on myoblasts, and the role of bioactive glass composite materials on myogenic differentiation is unknown. Herein, we report the effects of BGNs with different compositions (60Si-BGN, 80Si-BGN, 100Si-BGN) on the myogenic differentiation in C2C12 cells and in vivo skeletal tissue regeneration. The results showed that 80Si-BGN could efficiently promote the myogenic differentiation of C1C12 cells, including the myotube formation and myogenic gene expression. The in vivo experiment in a rat skeletal muscle defect model also confirmed that 80Si-BGN could significantly improve the complete regeneration of skeletal muscle tissue during 4 weeks implantation. This work firstly demonstrated evidence that BGN could be the bioactive material in enhancing skeletal muscle regeneration.

Latest developments in engineered skeletal muscle tissues for drug discovery and development

ABSTRACT Introduction With the advances in skeletal muscle tissue engineering, new platforms have arisen with important applications in biology studies, disease modeling, and drug testing. Current developments highlight the quest for engineering skeletal muscle tissues with higher complexity . These new human skeletal muscle tissue models will be powerful tools for drug discovery and development and disease modeling. Areas covered The authors review the latest advances in in vitro models of engineered skeletal muscle tissues used for testing drugs with a focus on the use of four main cell culture techniques: Cell cultures in well plates, in microfluidics, in organoids, and in bioprinted constructs. Additional information is provided on the satellite cell niche. Expert opinion In recent years, more sophisticated in vitro models of skeletal muscle tissues have been fabricated. Important developments have been made in stem cell research and in the engineering of human skeletal muscle tissue. Some platforms have already started to be used for drug testing, notably those based on the parameters of hypertrophy/atrophy and the contractibility of myotubes. More developments are expected through the use of multicellular types and multi-materials as matrices . The validation and use of these models in drug testing should now increase.