Skeletal Muscle Tissue Engineering Research Articles

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.

Conductive self-healing biodegradable hydrogel based on hyaluronic acid-grafted-polyaniline as cell recruitment niches and cell delivery carrier for myogenic differentiation and skeletal muscle regeneration

Scaffold is one of the most important roles in skeletal muscle tissue engineering. Being capable of mimicking extracellular matrix components, having electrical conductivity, and recruiting cells proactively to meet the cell-free requests are the proper characteristics of tissue engineering scaffolds. In this work, a series of leucine (Leu) loaded self-healing conductive hydrogels based on aminated hyaluronic acid-graft-polyaniline (AHA-PANI) and oxidized hyaluronic (OHA) were prepared for skeletal muscle tissue engineering. Leu can promote skeletal myogenesis and hydrogel can provide a suitable growth environment for cells. They have self-healing properties, antioxidant activity, suitable modulus, good electrical conductivity, high porosity, and proper swelling ratio. Additionally, these hydrogels have good cytocompatibility and can provide a 3D culture environment for C2C12 and hADSC cells. The Leu-loaded hydrogel has recruiting ability of cells and can promote myogenic differentiation of C2C12 cells. Besides, these hydrogels enable sustained cell delivery, and have good in vivo degradability and exhibited promoting effect on skeletal muscle regeneration in the rat tibialis anterior muscle defect model with reduced post-repair inflammatory factor expression and enhanced myogenic differentiation-related gene expression. All the results indicated that these conductive hydrogels as self-healing cell recruitment niches are excellent biomaterial for the treatment of volumetric muscle loss injury.

Pro-Myogenic Environment Promoted by the Synergistic Effect of Conductive Polymer Nanocomposites Combined with Extracellular Zinc Ions.

A new strategy based on the combination of electrically conductive polymer nanocomposites and extracellular Zn2+ ions as a myogenic factor was developed to assess its ability to synergically stimulate myogenic cell response. The conductive nanocomposite was prepared with a polymeric matrix and a small amount of graphene (G) nanosheets (0.7% wt/wt) as conductive filler to produce an electrically conductive surface. The nanocomposites' surface electrical conductivity presented values in the range of human skeletal muscle tissue. The biological evaluation of the cell environment created by the combination of the conductive surface and extracellular Zn2+ ions showed no cytotoxicity and good cell adhesion (murine C2C12 myoblasts). Amazingly, the combined strategy, cell-material interface with conductive properties and Zn bioactive ions, was found to have a pronounced synergistic effect on myoblast proliferation and the early stages of differentiation. The ratio of differentiated myoblasts cultured on the conductive nanocomposites with extracellular Zn2+ ions added in the differentiation medium (serum-deprived medium) was enhanced by more than 170% over that of non-conductive surfaces (only the polymeric matrix), and more than 120% over both conductive substrates (without extracellular Zn2+ ions) and non-conductive substrates with extracellular Zn2+. This synergistic effect was also found to increase myotube density, myotube area and diameter, and multinucleated myotube formation. MyoD-1 gene expression was also enhanced, indicating the positive effect in the early stages of myogenic differentiation. These results demonstrate the great potential of this combined strategy, which stands outs for its simplicity and robustness, for skeletal muscle tissue engineering applications.

Vascularization of Poly-ε-Caprolactone-Collagen I-Nanofibers with or without Sacrificial Fibers in the Neurotized Arteriovenous Loop Model

Electrospun nanofibers represent an ideal matrix for the purpose of skeletal muscle tissue engineering due to their highly aligned structure in the nanoscale, mimicking the extracellular matrix of skeletal muscle. However, they often consist of high-density packed fibers, which might impair vascularization. The integration of polyethylene oxide (PEO) sacrificial fibers, which dissolve in water, enables the creation of less dense structures. This study examines potential benefits of poly-ε-caprolactone-collagen I-PEO-nanoscaffolds (PCP) in terms of neovascularization and distribution of newly formed vessels compared to poly-ε-caprolactone -collagen I-nanoscaffolds (PC) in a modified arteriovenous loop model in the rat. For this purpose, the superficial inferior epigastric artery and vein as well as a motor nerve branch were integrated into a multilayer three-dimensional nanofiber scaffold construct, which was enclosed by an isolation chamber. Numbers and spatial distribution of sprouting vessels as well as macrophages were analyzed via immunohistochemistry after two and four weeks of implantation. After four weeks, aligned PC showed a higher number of newly formed vessels, regardless of the compartments formed in PCP by the removal of sacrificial fibers. Both groups showed cell influx and no difference in macrophage invasion. In this study, a model of combined axial vascularization and neurotization of a PCL-collagen I-nanofiber construct could be established for the first time. These results provide a foundation for the in vivo implantation of cells, taking a major step towards the generation of functional skeletal muscle tissue.

Extrusion 3D (Bio)Printing of Alginate-Gelatin-Based Composite Scaffolds for Skeletal Muscle Tissue Engineering.

Volumetric muscle loss (VML), which involves the loss of a substantial portion of muscle tissue, is one of the most serious acute skeletal muscle injuries in the military and civilian communities. The injured area in VML may be so severely affected that the body loses its innate capacity to regenerate new functional muscles. State-of-the-art biofabrication methods such as bioprinting provide the ability to develop cell-laden scaffolds that could significantly expedite tissue regeneration. Bioprinted cell-laden scaffolds can mimic the extracellular matrix and provide a bioactive environment wherein cells can spread, proliferate, and differentiate, leading to new skeletal muscle tissue regeneration at the defect site. In this study, we engineered alginate-gelatin composite inks that could be used as bioinks. Then, we used the inks in an extrusion printing method to develop design-specific scaffolds for potential VML treatment. Alginate concentration was varied between 4-12% w/v, while the gelatin concentration was maintained at 6% w/v. Rheological analysis indicated that the alginate-gelatin inks containing 12% w/v alginate and 6% w/v gelatin were most suitable for developing high-resolution scaffolds with good structural fidelity. The printing pressure and speed appeared to influence the printing accuracy of the resulting scaffolds significantly. All the hydrogel inks exhibited shear thinning properties and acceptable viscosities, though 8-12% w/v alginate inks displayed properties ideal for printing and cell proliferation. Alginate content, crosslinking concentration, and duration played significant roles (p < 0.05) in influencing the scaffolds' stiffness. Alginate scaffolds (12% w/v) crosslinked with 300, 400, or 500 mM calcium chloride (CaCl2) for 15 min yielded stiffness values in the range of 45-50 kPa, i.e., similar to skeletal muscle. The ionic strength of the crosslinking concentration and the alginate content significantly (p < 0.05) affected the swelling and degradation behavior of the scaffolds. Higher crosslinking concentration and alginate loading enhanced the swelling capacity and decreased the degradation kinetics of the printed scaffolds. Optimal CaCl2 crosslinking concentration (500 mM) and alginate content (12% w/v) led to high swelling (70%) and low degradation rates (28%) of the scaffolds. Overall, the results indicate that 12% w/v alginate and 6% w/v gelatin hydrogel inks are suitable as bioinks, and the printed scaffolds hold good potential for treating skeletal muscle defects such as VML.

Brush-Induced Orientation of Collagen Fibers in Layer-by-Layer Nanofilms: A Simple Method for the Development of Human Muscle Fibers.

The engineering of skeletal muscle tissue, a highly organized structure of myotubes, is promising for the treatment of muscle injuries and muscle diseases, for replacement, or for pharmacology research. Muscle tissue development involves differentiation of myoblasts into myotubes with parallel orientation, to ultimately form aligned myofibers, which is challenging to achieve on flat surfaces. In this work, we designed hydrogen-bonded tannic acid/collagen layer-by-layer (TA/COL LbL) nanofilms using a simple brushing method to address this issue. In comparison to films obtained by dipping, brushed TA/COL films showed oriented COL fibers of 60 nm diameter along the brushing direction. Built at acidic pH due to COL solubility, TA/COL films released TA in physiological conditions with a minor loss of thickness. After characterization of COL fibers' orientation, human myoblasts (C25CL48) were seeded on the oriented TA/COL film, ended by COL. After 12 days in a differentiation medium without any other supplement, human myoblasts were able to align on brushed TA/COL films and to differentiate into long aligned myotubes (from hundreds of μm up to 1.7 mm length) thanks to two distinct properties: (i) the orientation of COL fibers guiding myoblasts' alignment and (ii) the TA release favoring the differentiation. This simple and potent brushing process allows the development of anisotropic tissues in vitro which can be used for studies of drug discovery and screening or the replacement of damaged tissue.

Engineered skeletal muscle recapitulates human muscle development, regeneration and dystrophy.

Human pluripotent stem cell-derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for the derivation of skeletal muscle cells and their utility in skeletal muscle tissue engineering with the aim to model skeletal muscle regeneration and dystrophy in vitro. Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors followed by primary and secondary foetal myogenesis into three-dimensional muscle. To simulate Duchenne muscular dystrophy (DMD), a patient-specific induced pluripotent stem cell line was compared to a CRISPR/Cas9-edited isogenic control line. The established skeletal muscle differentiation protocol robustly and faithfully recapitulates critical steps of embryonic myogenesis in two-dimensional and three-dimensional cultures, resulting in functional human skeletal muscle organoids (SMOs) and engineered skeletal muscles (ESMs) with a regeneration-competent satellite-like cell pool. Tissue-engineered muscle exhibits organotypic maturation and function (up to 5.7±0.5mN tetanic twitch tension at 100Hz in ESM). Contractile performance could be further enhanced by timed thyroid hormone treatment, increasing the speed of contraction (time to peak contraction) as well as relaxation (time to 50% relaxation) of single twitches from 107±2 to 75±4ms (P<0.05) and from 146±6 to 100±6ms (P<0.05), respectively. Satellite-like cells could be documented as largely quiescent PAX7+ cells (75±6% Ki67- ) located adjacent to muscle fibres confined under a laminin-containing basal membrane. Activation of the engineered satellite-like cell niche was documented in a cardiotoxin injury model with marked recovery of contractility to 57±8% of the pre-injury force 21days post-injury (P<0.05 compared to Day 2 post-injury), which was completely blocked by preceding irradiation. Absence of dystrophin in DMD ESM caused a marked reduction of contractile force (-35±7%, P<0.05) and impaired expression of fast myosin isoforms resulting in prolonged contraction (175±14ms, P<0.05 vs. gene-edited control) and relaxation (238±22ms, P<0.05 vs. gene-edited control) times. Restoration of dystrophin levels by gene editing rescued the DMD phenotype in ESM. We introduce human muscle models with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease and repair.

Stem cell-based strategies for skeletal muscle tissue engineering.

Skeletal muscle tissue engineering has been a key area of focus over the years and has been of interest for developing regenerative strategies for injured or degenerative skeletal muscle tissue. Stem cells have gained increased attention as sources for developing skeletal muscle tissue for subsequent studies or potential treatments. Focus has been placed on understanding the molecular pathways that govern skeletal muscle formation in development to advance differentiation of stem cells towards skeletal muscle fates in vitro. Use of growth factors and transcription factors have long been the method for guiding skeletal muscle differentiation in vitro. However, further research in small molecule induced differentiation offers a xeno-free option that could result from use of animal derived factors.

Efficient co-isolation of microvascular endothelial cells and satellite cell-derived myoblasts from human skeletal muscle.

Vascularization of tissue-engineered constructs remains a key challenge in the field of skeletal muscle tissue engineering. One strategy for vascularizing organoids is in vitro pre-vascularization, relying on de novo assembly of undifferentiated endothelial cells into capillaries, a process termed vasculogenesis. In most endothelial cell research to date, human umbilical vein endothelial cells have been used primarily because of their availability. Nevertheless, this endothelial cell type is naturally not occurring in skeletal muscle tissue. Since endothelial cells display a tissue-specific phenotype, it is of interest to use muscle-specific microvascular endothelial cells to study pre-vascularization in skeletal muscle tissue engineering research. Thus far, tissue biopsies had to be processed in two separate protocols to obtain cells from the myogenic and the endothelial compartment. Here, we describe a novel, detailed protocol for the co-isolation of human skeletal muscle microvascular endothelial cells and satellite cell-derived myoblasts. It incorporates an automated mechanical and enzymatic tissue dissociation followed by magnetically activated cell sorting based on a combination of endothelial and skeletal muscle cell markers. Qualitative, quantitative, and functional characterization of the obtained cells is described and demonstrated by representative results. The simultaneous isolation of both cell types from the same donor is advantageous in terms of time efficiency. In addition, it may be the only possible method to isolate both cell types as the amount of tissue biopsy is often limited. The isolation of the two cell types is crucial for further studies to elucidate cell crosstalk in health and disease. Furthermore, the use of muscle-specific microvascular endothelial cells allows a shift towards engineering more physiologically relevant functional tissue, with downstream applications including drug screening and regenerative medicine.

Xeno-free bioengineered human skeletal muscle tissue using human platelet lysate-based hydrogels

Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate (PL)-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human PL reinforced with aldehyde-cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications.

Therapeutic Sources of Skeletal Muscle Regeneration from Volumetric Muscle Loss: A Narrative Review

PURPOSE: Minor skeletal muscle injuries can be repaired, but more extensive volumetric muscle loss (VML) leads to a permanent functional disability with ambiguous therapeutic outcomes, and reconstructive surgical procedures are constrained by donor tissue scarcity. This review assessed the considerable attention paid to biomaterials in healing damaged skeletal muscle.METHODS: A comprehensive search in PubMed, Web of Science, Google Scholar, and Wiley Online Library was conducted to obtain previous studies exploring the state of biocompatible tissue scaffolds for VML recovery.RESULTS: By regenerating the function of damaged skeletal muscle, tissue-engineered skeletal muscle construction could revolutionize the treatment of VML. However, transporting cells into the wounded muscle location presents a significant challenge because it may result in unfavorable immunological reactions. The development and validation of several biomaterials with varying physical and chemical natures to treat various muscle injuries have recently been undertaken to overcome this problem. This review discusses the relative benefits of satellite cells (SC), the most prevalent skeletal muscle stem cells employed to seed scaffolds.CONCLUSIONS: Biomaterials can be used with skeletal muscle stem cells and growth factors to repair VML because of their customizable and desirable physicochemical qualities. Owing to the capacity of SCs for self-renewal and their undifferentiated state, these cells are excellent candidates for cell therapy. A large gap exists between understanding SC behavior and how it can be used to repair and regenerate human skeletal muscle tissue. Thus, this review sought to portray the current knowledge on the lifespan of SCs and their involvement in exercise-induced muscle regeneration and hypertrophy.

NNOS-derived NO modulates force production and iNO-derived NO the excitability in C2C12-derived 3D tissue engineering skeletal muscle via different NO signaling pathways.

Nitric oxide (NO) is a bioactive gas produced by one of the three NO synthases: neuronal NOS (nNOS), inducible (iNOS), and endothelial NOS (eNOS). NO has a relevant modulatory role in muscle contraction; this takes place through two major signaling pathways: (i) activation of soluble guanylate cyclase and, thus, protein kinase G or (ii) nitrosylation of sulfur groups of cysteine. Although it has been suggested that nNOS-derived NO is the responsible isoform in muscle contraction, the roles of eNOS and iNOS and their signaling pathways have not yet been clarified. To elucidate the action of each pathway, we optimized the generation of myooids, an engineered skeletal muscle tissue based on the C2C12 cell line. In comparison with diaphragm strips from wild-type mice, 180 myooids were analyzed, which expressed all relevant excitation–contraction coupling proteins and both nNOS and iNOS isoforms. Along with the biochemical results, myooids treated with NO donor (SNAP) and unspecific NOS blocker (L-NAME) revealed a comparable NO modulatory effect on force production as was observed in the diaphragm strips. Under the effects of pharmacological tools, we analyzed the myooids in response to electrical stimulation of two possible signaling pathways and NO sources. The nNOS-derived NO exerted its negative effect on force production via the sGG-PKG pathway, while iNOS-derived NO increased the excitability in response to sub-threshold electrical stimulation. These results strengthen the hypotheses of previous reports on the mechanism of action of NO during force production, showed a novel function of iNOS-derived NO, and establish the myooid as a novel and robust alternative model for pathophysiological skeletal muscle research.

Polysaccharides, as biological macromolecule-based platforms in skeletal muscle tissue engineering: a systematic review

Traumatic injuries to skeletal muscles caused by accidents, surgeries, and strains result in a significant disability. Currently, the golden standard of treatment for skeletal muscle injuries is tissue grafting. However, this treatment strategy is associated with several challenges. Therefore, developing alternative strategies is highly demanded. In this context, skeletal muscle tissue engineering (SMTE) aims to regenerate defective muscle tissue using combinations of cells, growth factors, and biomaterials. Although significant progress has been made in SMTE, the inherent complexity of skeletal muscle tissue requires a versatile approach to develop scaffolds with high similarity to native muscle tissue. These scaffolds can be produced using synthetic or natural polymers. Among different biomaterial options, polysaccharides are ideal scaffolding platforms with potential applicability in different biomedical fields, such as drug delivery, guided tissue regeneration, and driving stem cells fate. Due to their versatile properties, biocompatibility, low cost, high availability, low immunogenicity, surface modification capabilities, and processability, the appeal of polysaccharides in SMTE is on the rise. Although different polysaccharides can be potentially used in SMTE, only alginate, chitosan, cellulose, and hyaluronic acid have been studied. In the current review, the recent progress, applications, and challenges of polysaccharides in SMTE will be highlighted.

Self‐actuating multilayer scaffold for skeletal muscle tissue engineering

AbstractElectroactive polymers (EAP) can alter and change their shape when subjected to an electric field, they have been investigated for a variety of purposes including smart drug delivery and artificial muscles. However, approaches to design electroactive hydrogel structures can be hindered by low polymeric conductivity, which then requires high electrical input to induce movement and results in low cellular viability. Our purpose in this study, was to reduce the input voltage required for movement by layering electroactive PEGDA:Acrylic acid (PEGDA:AA) with poly(ethylene glycol) diacrylate (PEGDA) modified with a conductive nanocomposite of colloidal poly(3,4 ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) and graphene oxide (GO). In this study PEDOT:PSS/GO was a flexible electrode that stimulated the PEGDA:AA. The multilayered hydrogel was evaluated for angular movement, electrical properties, and biocompatibility with C2C12 myoblast cells. The data showed that increasing the PEDOT:PSS/GO in the hydrogel decreased the conductivity, potentially due to the presence of GO. The addition of PEDOT:PSS/GO increased scaffold movement compared with PEGDA:AA alone when stimulated at 1 V. Fewer scaffolds displayed significantly more movement with increased voltage, but the 10% PEDOT:PSS/GO group experienced more movement at each tested voltage. C2C12 cells on the 10% PEDOT:PSS/GO were more metabolically active than cells on scaffolds with increased concentrations of PEDOT:PSS/GO. This work is an initial step in creating biocompatible scaffolds capable of actuation for tissue engineering.

Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research.

Skeletal muscle is now regarded as an endocrine organ based on its secretion of myokines and exerkines, which, in response to metabolic stimuli, regulate the crosstalk between the skeletal muscle and other metabolic organs in terms of systemic energy homeostasis. This conceptual basis of skeletal muscle as a metabolically active organ has provided insights into the potential role of physical inactivity and conditions altering muscle quality and quantity in the development of multiple metabolic disorders, including insulin resistance, obesity, and diabetes. Therefore, it is important to understand human muscle physiology more deeply in relation to the pathophysiology of metabolic diseases. Since monolayer cell lines or animal models used in conventional research differ from the pathophysiological features of the human body, there is increasing need for more physiologically relevant in vitro models of human skeletal muscle. Here, we introduce recent studies on in vitro models of human skeletal muscle generated from adult myogenic progenitors or pluripotent stem cells and summarize recent progress in the development of three-dimensional (3D) bioartificial muscle, which mimics the physiological complexity of native skeletal muscle tissue in terms of maturation and functionality. We then discuss the future of skeletal muscle 3D-organoid culture technology in the field of metabolic research for studying pathological mechanisms and developing personalized therapeutic strategies.

Wet Electrospinning and its Applications: A Review

In wet electrospinning, a natural or synthetic polymer solution is deposited on a non-solvent liquid coagulant used as collector. This technique can create 3D nanofiber scaffolds with better properties (e.g., porosity and high surface area) than those of traditional 2D scaffolds produced by standard electrospinning. Thanks to these characteristics, wet electrospinning can be employed in a wide range of tissue engineering and industrial applications. This review aims to broaden the panorama of this technique, its possible fields of action, and its range of common materials. Moreover, we also discuss its future trends. In this study, we review papers on this method published between 2017 and 2021 to establish the state of the art of wet electrospinning and its most important applications in cardiac, cartilage, hepatic, wound dressing, skin, neural, bone, and skeletal muscle tissue engineering. Additionally, we examine its industrial applications in water purification, air filters, energy, biomedical sensors, and textiles. The main results of this review indicate that 3D scaffolds for tissue engineering applications are biocompatible; mimic the extracellular matrix (ECM); allow stem cell viability and differentiation; and have high porosity, which provides greater cell infiltration compared to 2D scaffolds. Finally, we found that, in industrial applications of wet electrospinning: (1) additives improve the performance of pure polymers; (2) the concentration of the solution influences porosity and fiber packing; (3) flow rate, voltage, and distance modify fiber morphology; (4) the surface tension of the non-solvent coagulant on which the fibers are deposited has an effect on their porosity, compaction, and mechanical properties; and (5) deposition time defines scaffold thickness.&#x0D;

Enhancing Myoblast Fusion and Myotube Diameter in Human 3D Skeletal Muscle Constructs by Electromagnetic Stimulation.

Skeletal muscle tissue engineering (SMTE) aims at the in vitro generation of 3D skeletal muscle engineered constructs which mimic the native muscle structure and function. Although native skeletal muscle is a highly dynamic tissue, most research approaches still focus on static cell culture methods, while research on stimulation protocols indicates a positive effect, especially on myogenesis. A more mature muscle construct may be needed especially for the potential applications for regenerative medicine purposes, disease or drug disposition models. Most efforts towards dynamic cell or tissue culture methods have been geared towards mechanical or electrical stimulation or a combination of those. In the context of dynamic methods, pulsed electromagnetic field (PEMF) stimulation has been extensively used in bone tissue engineering, but the impact of PEMF on skeletal muscle development is poorly explored. Here, we evaluated the effects of PEMF stimulation on human skeletal muscle cells both in 2D and 3D experiments. First, PEMF was applied on 2D cultures of human myoblasts during differentiation. In 2D, enhanced myogenesis was observed, as evidenced by an increased myotube diameter and fusion index. Second, 2D results were translated towards 3D bioartificial muscles (BAMs). BAMs were subjected to PEMF for varying exposure times, where a 2-h daily stimulation was found to be effective in enhancing 3D myotube formation. Third, applying this protocol for the entire 16-days culture period was compared to a stimulation starting at day 8, once the myotubes were formed. The latter was found to result in significantly higher myotube diameter, fusion index, and increased myosin heavy chain 1 expression. This work shows the potential of electromagnetic stimulation for enhancing myotube formation both in 2D and 3D, warranting its further consideration in dynamic culturing techniques.

3D bioprinting of gellan gum-based hydrogels tethered with laminin-derived peptides for improved cellular behavior.

The treatment of skeletal muscle defects is still a topic of noteworthy concern since surgical intervention is not capable of recovering muscle function. Herein, we propose myoblasts laden in laminin-inspired biofunctionalized gellan gum hydrogels as promising tissue-engineered skeletal muscle surrogates. Gellan gum-based hydrogels were developed by combining native gellan gum (GG) and GG tethered with laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T) or RKRLQVQLSIRTC (Q)), using different polymer content (0.75%-1.875%). Hydrogels were characterized in terms of compressive modulus, molecules trafficking, and C2C12 adhesion. Hydrogels with higher polymeric content (1.125%-1.875%) showed higher stiffness whereas hydrogels with lower polymer content (0.75%-1.125%) showed higher fluorescein isothiocyanate-dextran molecules diffusion. Cell spreading was achieved regardless of the laminin-derived peptide but preferred in hydrogels with higher polymer content (1.125%-1.875%). Taken together, hydrogels with 1.125% of polymer content were selected for printability analysis. GG-based inks showed a non-newtonian, shear-thinning, and thixotropic behavior suitable for printing. Accordingly, all inks were printable, but inks tethered with T and Q peptides presented some signs of clogging. Cell viability was affected after printing but increased after 7 days of culture. After 7 days, cells were spreading but not showing significant signs of cell-cell communications. Therefore, cell density was increased, thus, myocytes loaded in V-tethered GG-based inks showed higher cell-cell communication, spreading morphology, and alignment 7, 14 days post-printing. Overall, myoblasts laden in laminin-inspired biofunctionalized GG-based hydrogels are a promising skeletal muscle surrogate with the potential to be used as in vitro model or explored for further in vivo applications.

Changes in Elastic Moduli of Fibrin Hydrogels Within the Myogenic Range Alter Behavior of Murine C2C12 and Human C25 Myoblasts Differently.

Fibrin hydrogels have proven highly suitable scaffold materials for skeletal muscle tissue engineering in the past. Certain parameters of those types of scaffolds, however, greatly affect cellular mechanobiology and therefore the myogenic outcome. The aim of this study was to identify the influence of apparent elastic properties of fibrin scaffolds in 2D and 3D on myoblasts and evaluate if those effects differ between murine and human cells. Therefore, myoblasts were cultured on fibrin-coated multiwell plates (“2D”) or embedded in fibrin hydrogels (“3D”) with different elastic moduli. Firstly, we established an almost linear correlation between hydrogels’ fibrinogen concentrations and apparent elastic moduli in the range of 7.5 mg/ml to 30 mg/ml fibrinogen (corresponds to a range of 7.7–30.9 kPa). The effects of fibrin hydrogel elastic modulus on myoblast proliferation changed depending on culture type (2D vs 3D) with an inhibitory effect at higher fibrinogen concentrations in 3D gels and vice versa in 2D. The opposite effect was evident in differentiating myoblasts as shown by gene expression analysis of myogenesis marker genes and altered myotube morphology. Furthermore, culture in a 3D environment slowed down proliferation compared to 2D, with a significantly more pronounced effect on human myoblasts. Differentiation potential was also substantially impaired upon incorporation into 3D gels in human, but not in murine, myoblasts. With this study, we gained further insight in the influence of apparent elastic modulus and culture type on cellular behavior and myogenic outcome of skeletal muscle tissue engineering approaches. Furthermore, the results highlight the need to adapt parameters of 3D culture setups established for murine cells when applied to human cells.

Biomedical applications of three-dimensional bioprinted craniofacial tissue engineering.

Anatomical complications of the craniofacial regions often present considerable challenges to the surgical repair or replacement of the damaged tissues. Surgical repair has its own set of limitations, including scarcity of the donor tissues, immune rejection, use of immune suppressors followed by the surgery, and restriction in restoring the natural aesthetic appeal. Rapid advancement in the field of biomaterials, cell biology, and engineering has helped scientists to create cellularized skeletal muscle-like structures. However, the existing method still has limitations in building large, highly vascular tissue with clinical application. With the advance in the three-dimensional (3D) bioprinting technique, scientists and clinicians now can produce the functional implants of skeletal muscles and bones that are more patient-specific with the perfect match to the architecture of their craniofacial defects. Craniofacial tissue regeneration using 3D bioprinting can manage and eliminate the restrictions of the surgical transplant from the donor site. The concept of creating the new functional tissue, exactly mimicking the anatomical and physiological function of the damaged tissue, looks highly attractive. This is crucial to reduce the donor site morbidity and retain the esthetics. 3D bioprinting can integrate all three essential components of tissue engineering, that is, rehabilitation, reconstruction, and regeneration of the lost craniofacial tissues. Such integration essentially helps to develop the patient-specific treatment plans and damage site-driven creation of the functional implants for the craniofacial defects. This article is the bird's eye view on the latest development and application of 3D bioprinting in the regeneration of the skeletal muscle tissues and their application in restoring the functional abilities of the damaged craniofacial tissue. We also discussed current challenges in craniofacial bone vascularization and gave our view on the future direction, including establishing the interactions between tissue-engineered skeletal muscle and the peripheral nervous system.