Muscle Tissue Engineering Research Articles

Piezoelectric nanocomposite bioink and ultrasound stimulation modulate early skeletal myogenesis.

Despite the significant progress in bioprinting for skeletal muscle tissue engineering, new stimuli-responsive bioinks to boost the myogenesis process are highly desirable. In this work, we developed a printable alginate/Pluronic-based bioink including piezoelectric barium titanate nanoparticles (nominal diameter: ∼60 nm) for the 3D bioprinting of muscle cell-laden hydrogels. The aim was to investigate the effects of the combination of piezoelectric nanoparticles with ultrasound stimulation on early myogenic differentiation of the printed structures. After the characterization of nanoparticles and bioinks, viability tests were carried out to investigate three nanoparticle concentrations (100, 250, and 500 μg mL-1) within the printed structures. An excellent cytocompatibility was confirmed for nanoparticle concentrations up to 250 μg mL-1. TEM imaging demonstrated the internalization of BTNPs in intracellular vesicles. The combination of piezoelectric nanoparticles and ultrasound stimulation upregulated the expression of MYOD1, MYOG, and MYH2 and enhanced cell aggregation, which is a crucial step for myoblast fusion, and the presence of MYOG in the nuclei. These results suggest that the direct piezoelectric effect induced by ultrasound on the internalized piezoelectric nanoparticles boosts myogenesis in its early phases.

Nanoengineered myogenic scaffolds for skeletal muscle tissue engineering.

Extreme loss of skeletal muscle overwhelms the natural regenerative capability of the body, results in permanent disability and substantial economic burden. Current surgical techniques result in poor healing, secondary injury to the autograft donor site, and incomplete recuperation of muscle function. Most current tissue engineering and regenerative strategies fail to create an adequate mechanical and biological environment that enables cell infiltration, proliferation, and myogenic differentiation. In this study, we present a nanoengineered skeletal muscle scaffold based on functionalized gelatin methacrylate (GelMA) hydrogel, optimized for muscle progenitors' proliferation and differentiation. The scaffold was capable of controlling the release of insulin-like growth factor 1 (IGF-1), an important myogenic growth factor, by utilizing the electrostatic interactions with LAPONITE® nanoclays (NCs). Physiologically relevant levels of IGF-1 were maintained during a controlled release over two weeks. The NC was able to retain 50% of the released IGF-1 within the hydrogel niche, significantly improving cellular proliferation and differentiation compared to control hydrogels. IGF-1 supplemented medium controls required 44% more IGF-1 than the comparable NC hydrogel composites. The nanofunctionalized scaffold is a viable option for the treatment of extreme muscle injuries and offers scalable benefits for translational interventions and the growing field of clean meat production.

A multi-functional SiO32--releasing hydrogel with bioinspired mechanical properties and biodegradability for vascularized skeletal muscle regeneration.

Vascularized skeletal muscle regeneration remains a great medical need but significant challenge. Biomaterial strategies that can facilitate the regeneration of muscle fibers and blood vessels are unavailable. Herein, we report a new cell- and drug-free biomaterial-based strategy for the repair of severely injured skeletal muscles. A novel multi-functional silicate ion-releasing hydrogel (SRH) was developed by dissolving PVA and starch in Na2SiO3 solutions, followed by freeze-thawing treatment. The mechanical properties and degradation profile of the SRH could be easily adjusted by altering the amylose/amylopectin ratio of starch. The SRH efficiently releases silicate ions to create a favorable microenvironment for enhanced skeletal muscle repair, while the mechanical properties and biodegradability of SRHs is adjusted to match the muscle regeneration environment. Silicate ions released from the SRH simultaneously promote myoblast proliferation and myogenic differentiation, decrease oxidative stress, and enhance the angiogenesis of vascular endothelial cells in vitro. Silicate ions released from the SRH scaffold with bioinspired mechanical properties and biodegradability promote the de novo formation of muscle fibers and blood vessels while inhibiting tissue fibrosis, leading to enhanced vascularized muscle regeneration in vivo. With multiple biofunctions and mechanical/degradation tunability, the SRH platform bears great potential in the skeletal muscle tissue engineering and treatment of formidable clinical problems such as volumetric muscle loss and sarcopenia.

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle

Systems for Muscle Cell Differentiation: From Bioengineering to Future Food.

In light of pressing issues, such as sustainability and climate change, future protein sources will increasingly turn from livestock to cell-based production and manufacturing activities. In the case of cell-based or cultured meat a relevant aspect would be the differentiation of muscle cells into mature muscle tissue, as well as how the microsystems that have been developed to date can be developed for larger-scale cultures. To delve into this aspect we review previous research that has been carried out on skeletal muscle tissue engineering and how various biological and physicochemical factors, mechanical and electrical stimuli, affect muscle cell differentiation on an experimental scale. Material aspects such as the different biomaterials used and 3D vs. 2D configurations in the context of muscle cell differentiation will also be discussed. Finally, the ability to translate these systems to more scalable bioreactor configurations and eventually bring them to a commercial scale will be touched upon.

Homemade bread: Repurposing an ancient technology for in vitro tissue engineering

Numerous biomaterial scaffolds have been developed which provide architectures to support the proliferation of mammalian cells. Scaffolds derived from plant components have been utilized in several tissue engineering applications, including the production of cultured meats. Bread crumb is a common ingredient employed as a texturizer and filler in existing manufacturing processes for the production of animal meat products. Though an unconventional choice as a scaffolding material, we developed a yeast-free “soda bread” with controllable porosity and mechanical properties which is stable over several weeks in culture with fibroblasts, myoblasts and pre-osteoblasts. All cells were able to proliferate throughout the three-dimensional scaffolds, depositing extra-cellular matrix while exhibiting low stress and high viability. Importantly, myoblasts were also able to differentiate into myotubes, a key step required for the culture of skeletal muscle tissue. The results suggest opportunities for the dual-use possibility of utilizing existing texturizer and filler components in future lab grown meat products, however this will of course require further validation. Regardless, the bread-derived scaffolds presented here are simply produced, inherently edible and support muscle tissue engineering, qualities which highlight their utility in the production of future meat products.

In-vitro effectiveness of poly-β-alanine reinforced poly(3-hydroxybutyrate) fibrous scaffolds for skeletal muscle regeneration

In skeletal muscle tissue engineering, success has not been achieved yet, since the properties of the tissue cannot be fully mimicked. The aim of this study is to investigate the potential use of poly-3-hydroxybutyrate (P3HB)/poly-β-alanine (PBA) fibrous tissue scaffolds with piezoelectric properties for skeletal muscle regeneration. Random and aligned P3HB/PBA (5:1) fibrous matrices were prepared by electrospinning with average diameters of 951 ± 153 nm and 891 ± 247 nm, respectively. X-ray diffraction (XRD) analysis showed that PBA reinforcement and aligned orientation of fibers reduced the crystallinity and brittleness of P3HB matrix. While tensile strength and elastic modulus of random fibrous matrices were determined as 3.9 ± 1.0 MPa and 86.2 ± 10.6 MPa, respectively, in the case of aligned fibers they increased to 8.5 ± 1.8 MPa and 378.2 ± 4.2 MPa, respectively. Aligned matrices exhibited a soft and an elastic behaviour with ~70% elongation in similar to the natural tissue. For the first time, d33 piezoelectric modulus of P3HB/PBA matrices were measured as 5 pC/N and 5.3 pC/N, for random and aligned matrices, respectively. Cell culture studies were performed with C2C12 myoblastic cell line. Both of random and aligned P3HB/PBA fibrous matrices supported attachment and proliferation of myoblasts, but cells cultured on aligned fibers formed regular and thick myofibril structures similar to the native muscle tissue. Reverse transcription polymerase chain reaction (RT-qPCR) analysis indicated that MyoD gene was expressed in the cells cultured on both fiber orientation, however, on the aligned fibers significant increase was determined in Myogenin and Myosin Heavy Chain (MHC) gene expressions, which indicate functional tubular structures. The results of RT-qPCR analysis were also supported with immunohistochemistry for myogenic markers. These in vitro studies have shown that piezoelectric P3HB/PBA aligned fibrous scaffolds can successfully mimic skeletal muscle tissue with its superior chemical, morphological, mechanical, and electroactive properties.

Micropatterned gellan gum-based hydrogels tailored with laminin-derived peptides for skeletal muscle tissue engineering

The efficacy of current therapies for skeletal muscle disorders/injuries are limited urging the need for new treatments. Skeletal muscle tissue engineered platforms represent a promising tool to shed light on the pathophysiology of skeletal muscle disorders/injuries and to investigate the efficacy of new therapies. Herein, we developed a skeletal muscle platform composed of aligned and differentiated myoblasts on micropatterned gellan gum (GG)-based hydrogels tailored with a laminin-derived peptide. To this aim, the binding of murine skeletal muscle cells (C2C12) to different laminin-derived peptides (CIKVAVS (V), KNRLTIELEVRTC (T), and RKRLQVQLSIRTC (Q)) and the binding of laminin-derived peptides to chemically functionalized GG was studied. C2C12-binding to peptide V, T and Q was 10%, 48% and 25%, whereas the peptide tethering to GG was 60%, 40% and 31%, respectively. Peptide-biofunctionalized hydrogels prepared with different polymer content showed different mechanics and peptide exposure at hydrogel surface. Cellular adhesion was detected in all hydrogel formulations, but spreading and differentiation was only promoted in peptide Q-biofunctionalized hydrogels and preferably in stiffer hydrogels. Myoblast alignment was promoted in micropatterned hydrogel surfaces. Overall, the engineered skeletal muscle herein proposed can be further explored as a platform to better understand skeletal muscle disorders/injuries and to screen new therapies.

Fabrication and evaluation of homogeneous alginate/polyacrylamide–chitosan–gelatin composite hydrogel scaffolds based on the interpenetrating networks for tissue engineering

AbstractInterpenetrating polymer network (IPN) is generally considered to be one of the most effective methods for imparting high‐strength mechanical properties to hydrogel materials. To broaden the application of alginate hydrogel in skeletal muscle or muscle tissue engineering, the interpenetrating network technology and layer‐by‐layer (LBL) electrostatic assembly were proposed to fabricate the homogeneous alginate/polyacrylamide–chitosan–gelatin (Alg/PAAM–CS–GT) composite hydrogel scaffolds, using the hydroxyapatite/d‐glucono‐δ‐lactone (HAP/GDL) complex as the gelling system. The effects of different components of alginate/polyacrylamide (Alg/PAAM) on the morphology, mechanical properties, swelling, degradability, and cytocompatibility of the hydrogel scaffolds were comparatively characterized. Experimental results showed that the as‐prepared Alg/PAAM–CS–GT composite hydrogel scaffolds exhibited sharp edges and regular 3D structures. Their pore structure could be regulated by changing the content of PAAM in Alg/PAAM–CS–GT composite hydrogel scaffolds. And the filling of PAAM reduced the pores and thickened the pore walls, which obviously enhanced the mechanical properties of Alg/PAAM–CS–GT composite hydrogel scaffolds. FT‐IR and thermogravimetric analysis showed that O‧‧‧N hydrogen bonding between COOH of SA and NH2 of PAAM could be helpful to improve the thermal stability of the composite hydrogel scaffolds. The swelling and biodegradation measurements indicated that the swelling and degradation of Alg/PAAM–CS–GT composite hydrogel scaffolds could be effectively regulated by changing the component content of the composite hydrogel. In addition, the in vitro cytocompatibility analysis showed that the Alg/PAAM–CS–GT composite hydrogel scaffolds could support the growth of MC3T3‐E1 cells and promote cell proliferation and differentiation. According to the in vitro cytocompatibility test results, the optimum PAAM content for the Alg/PAAM–CS–GT composite hydrogel scaffolds was 60%. Because of good morphology, well‐developed pores, excellent mechanical properties, and high biological activity of Alg/PAAM–CS–GT composite hydrogel scaffolds, they could be suitable for application in the tissue engineering field.

3D Printed Biodegradable Polyurethaneurea Elastomer Recapitulates Skeletal Muscle Structure and Function.

Effective skeletal muscle tissue engineering relies on control over the scaffold architecture for providing muscle cells with the required directionality, together with a mechanical property match with the surrounding tissue. Although recent advances in 3D printing fulfill the first requirement, the available synthetic polymers either are too rigid or show unfavorable surface and degradation profiles for the latter. In addition, natural polymers that are generally used as hydrogels lack the required mechanical stability to withstand the forces exerted during muscle contraction. Therefore, one of the most important challenges in the 3D printing of soft and elastic tissues such as skeletal muscle is the limitation of the availability of elastic, durable, and biodegradable biomaterials. Herein, we have synthesized novel, biocompatible and biodegradable, elastomeric, segmented polyurethane and polyurethaneurea (TPU) copolymers which are amenable for 3D printing and show high elasticity, low modulus, controlled biodegradability, and improved wettability, compared to conventional polycaprolactone (PCL) and PCL-based TPUs. The degradation profile of the 3D printed TPU scaffold was in line with the potential tissue integration and scaffold replacement process. Even though TPU attracts macrophages in 2D configuration, its 3D printed form showed limited activated macrophage adhesion and induced muscle-like structure formation by C2C12 mouse myoblasts in vitro, while resulting in a significant increase in muscle regeneration in vivo in a tibialis anterior defect in a rat model. Effective muscle regeneration was confirmed with immunohistochemical assessment as well as evaluation of electrical activity produced by regenerated muscle by EMG analysis and its force generation via a custom-made force transducer. Micro-CT evaluation also revealed production of more muscle-like structures in the case of implantation of cell-laden 3D printed scaffolds. These results demonstrate that matching the tissue properties for a given application via use of tailor-made polymers can substantially contribute to the regenerative outcomes of 3D printed tissue engineering scaffolds.

3D Printing of Skeleton Muscle Tissue Engineering Scaffolds

The aim of skeletal muscle tissue engineering is to replace or repair skeletal muscle functions that have been injured or lost part of their functions. Skeletal muscle tissue engineering is an important strategy for muscle injury repair. The previous review on skeletal muscle tissue engineering was limited to the discussion of seed cells, biological materials and growth factors. In recent years, research results in this field have continued to emerge. This paper first briefly introduces the anatomy of skeletal muscle, and then combines the latest domestic and foreign study, from the basic problems of tissue engineering skeletal muscle construction to its vascularization, neuralization, etc., to review the current research status of skeletal muscle tissue engineering using three-dimensional (3D) printing, and look forward to its research prospects.

Umbilical Cord Mesenchymal Stem Cell Therapy for Regenerative Treatment of Rheumatoid Arthritis: Opportunities and Challenges.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology with a high rate of disability. Traditional treatments for RA remain a challenging issue. For example, nonsteroidal anti-inflammatory drugs (NSAIDs) have no therapeutic effects on joint destruction, and the prominent side effects include gastrointestinal symptoms. RA is characterized by recurrence and bone attrition. Therefore, regenerative medicine and the use of umbilical cord mesenchymal stem cell (UC-MSC) therapies have recently emerged as potential options. UC-MSCs are multifunctional stem cells that are present in neonatal umbilical cord tissue and can differentiate into many kinds of cells, which have broad clinical application prospects in the tissue engineering of bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium, and myocardium. Moreover, UC-MSCs have advantages, such as convenient collection of materials and no ethical disputes; thus, these cells have attracted increasing attention from researchers. However, there are few clinical studies regarding UC-MSC therapy for RA. In this paper, we will review traditional drugs for RA treatment and then focus on UC-MSC therapy for RA, including preclinical and clinical UC-MSC applications for RA patients in the context of regenerative medicine. Finally, we will summarize the challenges and perspectives of UC-MSCs as a potential therapeutic strategy for RA. This review will help to design and discover more potent and efficacious treatments for RA patients and aid in advancing this class of cell therapy.

Global Evolution of Skeletal Muscle Tissue Engineering: A Scientometric Research.

Skeletal muscle tissue engineering (SMTE) is of great significance in the study of skeletal muscle physiology and pathology, which could be used in skeletal muscle graft. The scientometric analysis of SMTE can help researchers to quickly understand the evolutive history, status, novelties, and trend of this field. In this study, we performed a scientometric study that can be used to construct and visualize networks of SMTE using VOSviewer. A total of 1384 documents published between 1994 and 2020 were retrieved and analyzed. Our results showed that number of publications in SMTE has increased slowly from 1994 to 2014 and has increased rapidly from 2015 to 2020. The geographical distribution of publications in terms of total publications about SMTE is concentrated in Europe and the United States. The most productive institution was University of Michigan, while Harvard University and the University of Pittsburgh were ranked the second and third places. SMTE influenced a wide spectrum of disciplines, including Biology and Medicine and Physical Sciences. In addition, the research hotspot of SMTE was expanding from seed cells to the combination with advanced strategies (electrostatic spinning, bioprinting, and materials) for emulating the highly bionic engineered skeletal muscle tissues. This study provided a unique perspective for understanding the history and trends of SMTE, which could help to promote the rapid development of the field. Impact statement Skeletal muscle tissue engineering (SMTE), which acts as an important branch of tissue engineering, hold a great promise in the study of skeletal muscle physiology and pathology. The field of SMTE has developed rapidly in recent decades while still lacking studies based on scientometric methods. This article provided the first scientometric study of SMTE from development trends and evolution of the field. The results indicated that the field of SMTE was experiencing rapid growth and had a significant impact on multiple fields, particularly in Biology and Medicine and Physical Sciences.

Myogenic Potential of Extracellular Matrix Derived from Decellularized Bovine Pericardium.

Skeletal muscles represent 40% of body mass and its native regenerative capacity can be permanently lost after a traumatic injury, congenital diseases, or tumor ablation. The absence of physiological regeneration can hinder muscle repair preventing normal muscle tissue functions. To date, tissue engineering (TE) represents one promising option for treating muscle injuries and wasting. In particular, hydrogels derived from the decellularized extracellular matrix (dECM) are widely investigated in tissue engineering applications thanks to their essential role in guiding muscle regeneration. In this work, the myogenic potential of dECM substrate, obtained from decellularized bovine pericardium (Tissuegraft Srl), was evaluated in vitro using C2C12 murine muscle cells. To assess myotubes formation, the width, length, and fusion indexes were measured during the differentiation time course. Additionally, the ability of dECM to support myogenesis was assessed by measuring the expression of specific myogenic markers: α-smooth muscle actin (α-sma), myogenin, and myosin heavy chain (MHC). The results obtained suggest that the dECM niche was able to support and enhance the myogenic potential of C2C12 cells in comparison of those grown on a plastic standard surface. Thus, the use of extracellular matrix proteins, as biomaterial supports, could represent a promising therapeutic strategy for skeletal muscle tissue engineering.

Regulation of Myogenic Differentiation by Topologically Microgrooved Surfaces for Skeletal Muscle Tissue Engineering.

Inspired by the natural topological structure of skeletal muscle tissue, the topological surface construction of bionic scaffolds for skeletal muscle repair has attracted great interest. Many previous studies have focused on the effects of the topological structure on myoblasts. However, these studies used only specific repeating sizes and shapes to achieve the myoblast alignment and myotube formation; moreover, the regulatory effects of the size of a topological structure on myogenic differentiation are often neglected, leading to a lack of guidance for the design of scaffolds for skeletal muscle tissue engineering. In this study, we fabricated a series of microgroove topographies with various widths and depths via a combination of soft lithography and melt-casting and studied their effects on the behaviors of skeletal muscle cells, especially myogenic differentiation, in detail. Microgrooved poly(lactic-co-glycolic acid) substrates were found to effectively regulate the proliferation, myogenic differentiation, and myotube formation of C2C12 cells, and the degree of myogenic differentiation was significantly dependent on signals in response to the size of the microgroove structure. Compared with their depth, the width of the microgroove structures can more strongly affect the myogenic differentiation of C2C12 cells, and the degree of myoblast differentiation was enhanced with increasing groove width. Microgroove structures with relatively large groove widths and small groove depths promoted the myogenic differentiation of C2C12 cells. In addition, the integrin-mediated focal adhesion kinase signaling pathway and MAPK signaling pathway were activated in cells in response to the external topological structure, and the size of the topological structure of the material surface effectively regulated the degree of the cellular response to the external topological structure. These results can guide the design of scaffolds for skeletal muscle tissue engineering and the construction of effective bionic scaffold surfaces for skeletal muscle regeneration.

Enhancing Peptide Biomaterials for Biofabrication.

Biofabrication using well-matched cell/materials systems provides unprecedented opportunities for dealing with human health issues where disease or injury overtake the body’s native regenerative abilities. Such opportunities can be enhanced through the development of biomaterials with cues that appropriately influence embedded cells into forming functional tissues and organs. In this context, biomaterials’ reliance on rigid biofabrication techniques needs to support the incorporation of a hierarchical mimicry of local and bulk biological cues that mimic the key functional components of native extracellular matrix. Advances in synthetic self-assembling peptide biomaterials promise to produce reproducible mimics of tissue-specific structures and may go some way in overcoming batch inconsistency issues of naturally sourced materials. Recent work in this area has demonstrated biofabrication with self-assembling peptide biomaterials with unique biofabrication technologies to support structural fidelity upon 3D patterning. The use of synthetic self-assembling peptide biomaterials is a growing field that has demonstrated applicability in dermal, intestinal, muscle, cancer and stem cell tissue engineering.

Electrohydrodynamic-direct-printed cell-laden microfibrous structure using alginate-based bioink for effective myotube formation

In this study, a fully aligned microfibrous structure fabricated using fibrin-assisted alginate bioink and electrohydrodynamic direct-printing was proposed for skeletal muscle tissue engineering. To safely construct the aligned alginate/fibrin microfibrous structure laden with myoblasts or endothelial cells, various printing conditions, such as an applied electric field, distance between the nozzle and target, and nozzle moving speed, were selected appropriately. Furthermore, to accelerate the formation of myotubes more efficiently, the alginate/fibrin bioink with vascular endothelial cells was co-printed into a spatially patterned structure within a myoblast-laden structure. The myoblast-laden structure co-cultured with endothelial cells presented fully aligned myotube formation and significantly greater myogenic differentiation compared to the myoblast-laden structure without the endothelial cells owing to the more abundant secretion of angiogenic cytokines. Also, when adipose stem cell- and endothelial cell-laden fibrous structure was implanted in a mouse volumetric muscle loss model, accelerated volumetric muscle repair was observed compared to the defect model. Based on the results, this study demonstrates an alginate-based bioink and new bio-fabricating method to obtain microfibrous cell-laden alginate/fibrin structures with mechanically stable and topographical cues. The proposed method can provide a myoblast/endothelial cell-laden fibrous alginate structure to efficiently induce engineering of skeletal muscle tissue, which could be used in muscle-on-a-chip or recovering structures of volumetric muscle defects.

3D anisotropic conductive fibers electrically stimulated myogenesis

Recapitulation of in vivo environments that drive muscle cells to organize into a physiologically relevant 3D architecture remains a major challenge for muscle tissue engineering. To recreate electrophysiology of muscle tissues, electroactive biomaterials have been used to stimulate muscle cells with exogenous electrical fields. In particular, the use of electroactive biomaterials with an anisotropic micro-/nanostructure that closely mimic the native skeletal-muscle extracellular matrix (ECM) is desirable for skeletal muscle tissue engineering. Herein, we present a hierarchically organized, anisotropic, and conductive Polycaprolactone/gold (PCL/Au) scaffold for guiding myoblasts alignment and promoting the elongation and maturation of myotubes under electrical stimulation. Culturing with H9c2 myoblasts cells indicated that the nanotopographic cues was crucial for nuclei alignment, while the presence of microscale grooves effectively enhanced both the formation and elongation of myotubes. The anisotropic structure also leads to anisotropic conductivity. Under electrical stimulation, the elongation and maturation of myotubes were significantly enhanced along the anisotropic scaffold. Specifically, compared to the unstimulated group (0 V), the myotube area percentage increased by 1.4, 1.9 and 2.4 times in the 1 V, 2 V, 3 V groups, respectively. In addition, the myotube average length in the 1 V group increased by 1.3 times compared to that of the unstimulated group, and significantly increased by 1.8 and 2.0 times in the 2 V, 3 V groups, respectively. Impressively, the longest myotubes reached more than 4 mm in both 2 V and 3 V groups. Overall, our conductive, anisotropic 3D nano/microfibrous scaffolds with the application of electrical stimulation provides a desirable platform for skeletal muscle tissue engineering.

Biomimetic 3D aligned conductive tubular cryogel scaffolds with mechanical anisotropy for 3D cell alignment, differentiation and in vivo skeletal muscle regeneration

Developing 3D conductive aligned cryogels has great potential for skeletal muscle trauma treatment because they can mimic anisotropic structure, conductivity, and recoverable cyclic compression of the microenvironment of native skeletal muscle. In this work, a series of cryogels possessing 3D aligned morphology, conductivity, and excellent anisotropic mechanical compression property based on gelatin (GT) and polydopamine coated carbon nanotubes (PCNTs) were fabricated as skeletal muscle tissue scaffolds by using unidirectional freeze casting technology. The aligned microstructure of cryogels depended on gelatin concentration, and GT7.5 (with the gelatin content of 7.5% w/v) showed excellent aligned structure. Interestingly, the mechanical property of the aligned cryogels was similar to that of native skeletal muscle in terms of the dynamic contraction behavior and the anisotropic compression property due to the internal anisotropy structure. The aligned cryogel GT7.5 with good biocompatibility significantly promoted the alignment and elongation of C2C12 myoblasts. Moreover, the introduction of PCNTs enhanced the mechanical properties of cryogel GT7.5 and had a positive effect on myogenic differentiation of C2C12 cells. The aligned conductive GT7.5C2 cryogel significantly promoted new born muscle tissue generation compared to non-aligned group (GT7.5C2N) and non-conductive group (GT7.5) in a rat tibialis anterior muscle defect model. These data suggested that the 3D aligned conductive cryogel with conductivity and anisotropic compression property is a promising scaffold candidate for skeletal muscle tissue engineering.