Skeletal Muscle Tissue Regeneration Research Articles

Engineering Nanoclusters of Cell Adhesive Ligands on Biomaterial Surfaces: Superior Cell Proliferation and Myotube Formation for Skeletal Muscle Tissue Regeneration.

Engineering biointerfaces with nanoscale clustering of integrin-binding cell adhesive peptides is critical for promoting receptor redistribution into signaling complexes. Skeletal muscle cells are exquisitely sensitive to integrin-mediated signaling, yet biomaterials supporting myogenesis through control of the density and nanodistribution of ligands have not been developed. Here, materials are developed with tailorable cell adhesive ligands distribution at the interface by independently controlling their global and local density to enhance myogenesis, by promoting myoblast growth and myotube formation. To this end, RGD-functionalized low-fouling polymer surfaces with global ligand densities (G) from 0-7µg peptide/mg polymer and average local ligand densities (L) from 1-6.3 ligands/cluster, are generated and characterized. Cell studies demonstrate improvements in cell adhesion, spreading, growth, and myotube formation up to a density of 7µg peptide/mg polymer with 4 ligands/cluster. Optimizing ligand density and distribution also promotes early myofiber maturation, identified by increased MF20 marker protein expression and sarcomere-forming myotubes. At higher ligand densities, these cell properties are decreased, indicating that ligand multivalency is a critical parameter for tailoring cell-material interactions, to a certain threshold. The findings provide new insights for designing next-generation biomaterials and hold promise for improved engineering of skeletal muscle.

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

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

Bioactive Hydrogels Inspired by Laminin: An Emerging Biomaterial for Tissue Engineering Applications.

Tissue or organ damage due to severe injuries or chronic diseases can adversely affect the quality of life. Current treatments rely on organ or tissue transplantation which has limitations including unavailability of donors, ethical issues, or immune rejection after transplantations. These limitations can be addressed by tissue regeneration which involves the development of bioactive scaffolds closely mimicking the extracellular matrix (ECM). One of the major components of ECM is the laminin protein which supports several tissues associated with important organs. In this direction, peptide-based hydrogels can effectively mimic the essential characteristics of laminin. While several reports have discussed the structure of laminin, the potential of laminin-derived peptide hydrogels as effective biomaterial for tissue engineering applications is yet to be discussed. In this context, the current review focuses on the structure of laminin and its role as an essential ECM protein. Further, the potential of short peptide hydrogels in mimicking the crucial properties of laminin is proposed. The review further highlights the significance of bioactive hydrogels inspired by laminin - in addressing numerous tissue engineering applications including angiogenesis, neural, skeletal muscle, liver, and adipose tissue regeneration along with a brief outlook on the future applications of these laminin-based hydrogels.

Exploration on pharmacological mechanisms of YZP against neuropathic pain via inhibiting spinal inflammation and the rationality of its compatibility

Ethnopharmacological relevanceYuanhu Zhitong Prescription (YZP) is a well-known traditional Chinese medicine (TCM) formula for neuropathic pain (NP) therapy with a satisfying clinical efficacy. However, the underlying pharmacological mechanism and its compatibility principle remain unclear. Aim of the studyThis study aims to investigate the analgesic and compatibility mechanisms of YZP on neuropathic pain (NP) at the gene and biological process levels. Materials and methodsThe chronic constriction injury (CCI) rats were intragastrically administrated with extracts of YZP, YH and BZ separately, and then mechanical hypersensitivity were measured to evaluate the analgesic effects between YH and BZ before and after compatibility. Then, RNA-seq and bioinformatics analyses were performed to elucidate the potential mechanisms underlying YZP's analgesia and compatibility. Finally, the expression levels and significant differences of key genes were analyzed. ResultsBehaviorally, both YZP and YH effectively alleviated mechanical allodynia in CCI rats, with YZP being superior to YH. In contrast, we did not observe an analgesic effect of BZ. Genetically, YZP, YH, and BZ reversed the expression levels of 52, 34, and 42 aberrant genes in the spinal cord of CCI rats, respectively. Mechanically, YZP was revealed to alleviate NP mainly by modulating the inflammatory response and neuropeptide signaling pathway, which are the dominant effective processes of YH. Interestingly, the effective targets of YZP were especially enriched in leukocyte activation and cytokine-mediated signaling pathways. Moreover, BZ was found to exert an adjunctive effect in enhancing the analgesic effect of YH by promoting skeletal muscle tissue regeneration and modulating calcium ion transport. ConclusionsYH, as the monarch drug, plays a dominant role in the analgesic effect of YZP that effectively relieves NP by inhibiting the spinal inflammation and neuropeptide signaling pathway. BZ, as the minister drug, not only synergistically enhances analgesic processes of YH but also helps to alleviate the accompanying symptoms of NP. Consequently, YZP exerted a more potent analgesic effect than YH and BZ alone. In conclusion, our findings offer new insights into understanding the pharmacological mechanism and compatibility principle of YZP, which may support its clinical application in NP therapy.

Bioactive Glass and Silica Particles for Skeletal and Cardiac Muscle Tissue Regeneration.

When skeletal and cardiac tissues are damaged, surgical approaches are not always successful and tissue regeneration approaches are investigated. Reports in the literature indicate that silica nanoparticles and bioactive glasses (BGs), including silicate bioactive glasses (e.g., 45S5 BG), phosphate glass fibers, boron-doped mesoporous BGs, borosilicate glasses, and aluminoborates, are promising for repairing skeletal muscle tissue. Silica nanoparticles and BGs have been combined with polymers to obtain aligned nanofibers and to maintain controlled delivery of nanoparticles for skeletal muscle repair. The literature indicates that cardiac muscle regeneration can be also triggered by the ionic products of BGs. This was observed to be due to the release of vascular endothelial growth factor and other growth factors from cardiomyocytes, which regulate endothelial cells to form capillary structures (angiogenesis). Specific studies, including both in vitro and in vivo approaches, are reviewed in this article. The analysis of the literature indicates that although the research field is still very limited, BGs are showing great promise for muscle tissue engineering and further research in the field should be carried out to expand our basic knowledge on the application of BGs in muscle (skeletal and cardiac) tissue regeneration. Impact statement This review highlights the potential of silica particles and bioactive glasses (BGs) for skeletal and cardiac tissue regeneration. These biomaterials create scaffolds triggering muscle cell differentiation. Ionic products from BGs stimulate growth factors, supporting angiogenesis in cardiac tissue repair. Further research is required to expand our know-how on silica particles and BGs in muscle tissue engineering.

Aligned Collagen Sponges with Tunable Pore Size for Skeletal Muscle Tissue Regeneration.

Volumetric muscle loss (VML) is a traumatic injury where at least 20% of the mass of a skeletal muscle has been destroyed and functionality is lost. The standard treatment for VML, autologous tissue transfer, is limited as approximately 1 in 10 grafts fail because of necrosis or infection. Tissue engineering strategies seek to develop scaffolds that can regenerate injured muscles and restore functionality. Many of these scaffolds, however, are limited in their ability to restore muscle functionality because of an inability to promote the alignment of regenerating myofibers. For aligned myofibers to form on a scaffold, myoblasts infiltrate the scaffold and receive topographical cues to direct targeted myofiber growth. We seek to determine the optimal pore size for myoblast infiltration and differentiation. We developed a method of tuning the pore size within collagen scaffolds while inducing longitudinal alignment of these pores. Significantly different pore sizes were generated by adjusting the freezing rate of the scaffolds. Scaffolds frozen at -20 °C contained the largest pores. These scaffolds promoted the greatest level of cell infiltration and orientation in the direction of pore alignment. Further research will be conducted to induce higher levels of myofiber formation, to ultimately create an off-the-shelf treatment for VML injuries.

Microscopic changes in experimentally damaged rabbit muscles under the influence of transplanted mesenchymal stem cells

The relevance of the work is related to the possibility of artificially adjusting the intensity of skeletal muscle tissue regeneration in animals, which will significantly affect the tactics of rehabilitation treatment of muscle injuries in the future. The purpose of the research was to determine the effectiveness of the transplanted mesenchymal stem cells on the recovery processes in experimentally injured rabbit muscles. The method of histological examination of microscopic changes in the experimentally damaged muscle tissue of the pelvic head of the biceps femoris muscle of rabbits was used. Microscopic studies of the regeneration process of experimentally damaged striated muscle tissue established that in animals after intramuscular injection of allogeneic mesenchymal stem cells, connective tissue developed around the defect on day 2 of the experiment. On day 4, a large number of stem cells were detected in the connective tissue at the site of muscle tissue damage. On day 6, only relatively small foci of muscle tissue regeneration were observed in this area. On day 8, only small focal accumulations of stem cells were detected in this location. On the 10th day after intramuscular injection of mesenchymal stem cells, newly developed muscle fibres appeared at the site of injury. On the 14th day after intramuscular injection of mesenchymal stem cells, fully developed muscle fibres and intermuscular connective tissue were observed at the site of injury. In rabbits, after intramuscular injection of allogeneic mesenchymal stem cells, all regenerative processes were more active than in animals of other experimental groups and were completely completed by day 14 of the experiment. The practical value of the results obtained is to clarify the patterns of development of reparative processes in the damaged muscle area and to determine the effectiveness of using transplanted allogeneic mesenchymal stem cells in stimulating recovery processes, which is important to consider in the treatment of muscle injuries using regenerative therapy

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.

Bioprinted anisotropic scaffolds with fast stress relaxation bioink for engineering 3D skeletal muscle and repairing volumetric muscle loss.

Viscoelastic hydrogels can enhance 3D cell migration and proliferation due to the faster stress relaxation promoting the arrangement of the cellular microenvironment. However, most synthetic photocurable hydrogels used as bioink materials for 3D bioprinting are typically elastic. Developing a photocurable hydrogel bioink with fast stress relaxation would be beneficial for 3D bioprinting engineered 3D skeletal muscles in vitro and repairing volumetric muscle loss (VML) in vivo; however, this remains an ongoing challenge. This study aims to develop an interpenetrating network (IPN) hydrogel with tunable stress relaxation using a combination of gelatin methacryloyl (GelMA) and fibrinogen. These IPN hydrogels with faster stress relaxation showed higher 3D cellular proliferation and better differentiation. A 3D anisotropic biomimetic scaffold was further developed via a printing gel-in-gel strategy, where the extrusion printing of cell-laden viscoelastic FG hydrogel within Carbopol supported gel. The 3D engineered skeletal muscle tissue was further developed via 3D aligned myotube formation and contraction. Furthermore, the cell-free 3D printed scaffold was implanted into a rat VML model, and both the short and long-term repair results demonstrated its ability to enhance functional skeletal muscle tissue regeneration. These data suggest that such viscoelastic hydrogel provided a suitable 3D microenvironment for enhancing 3D myogenic differentiation, and the 3D bioprinted anisotropic structure provided a 3D macroenvironment for myotube organization, which indicated the potential in skeletal muscle engineering and VML regeneration. STATEMENT OF SIGNIFICANCE: The development of a viscoelastic 3D aligned biomimetic skeletal muscle scaffold has been focused on skeletal muscle regeneration. However, a credible technique combining viscoelastic hydrogel and printing gel-in-gel strategy for fabricating skeletal muscle tissue was rarely reported. Therefore, in this study, we present an interpenetrating network (IPN) hydrogel with fast stress relaxation for 3D bioprinting engineered skeletal muscle via a printing gel-in-gel strategy. Such IPN hydrogels with tunable fast stress relaxation resulted in high 3D cellular proliferation and adequate differentiation in vitro. Besides, the 3D hydrogel-based scaffolds also enhance functional skeletal muscle regeneration in situ. We believe that this study provides several notable advances in tissue engineering that can be potentially used for skeletal muscle injury treatment in clinical.

Molecular Mechanisms Through Which Cannabidiol May Affect Skeletal Muscle Metabolism, Inflammation, Tissue Regeneration, and Anabolism: A Narrative Review.

Background: Cannabidiol (CBD), a nonintoxicating constituent of the cannabis plant, recently gained a lot of interest among athletes, since it is no longer considered as a prohibited substance by the World Anti-Doping Agency. The increasing prevalence of CBD use among athletes is driven by a perceived improvement in muscle recovery and a reduction in pain. However, compelling evidence from intervention studies is lacking and the precise mechanisms through which CBD may improve muscle recovery remain unknown. This highlights the need for more scientific studies and an evidence-based background. In the current review, the state-of-the-art knowledge on the effects of CBD on skeletal muscle tissue is summarized with special emphasis on the underlying mechanisms and molecular targets. More specifically, the large variety of receptor families that are believed to be involved in CBD's physiological effects are discussed. Furthermore, in vivo and in vitro studies that investigated the actual effects of CBD on skeletal muscle metabolism, inflammation, tissue regeneration, and anabolism are summarized, together with the functional effects of CBD supplementation on muscle recovery in human intervention trials. Overall, CBD was effective to increase the expression of metabolic regulators in muscle of obese mice (e.g., Akt, glycogen synthase kinase-3). CBD treatment in rodents reduced muscle inflammation following eccentric exercise (i.e., nuclear factor kappa B [NF-κB]), in a model of muscle dystrophy (e.g., interleukin-6, tumor necrosis factor alpha) and of obesity (e.g., COX-2, NF-κB). In addition, CBD did not affect in vitro or in vivo muscle anabolism, but improved satellite cell differentiation in dystrophic muscle. In humans, there are some indications that CBD supplementation improved muscle recovery (e.g., creatine kinase) and performance (e.g., squat performance). However, CBD doses were highly variable (between 16.7 and 150 mg) and there are some methodological concerns that should be considered. Conclusion: CBD has the prospective to become an adequate supplement that may improve muscle recovery. However, this research domain is still in its infancy and future studies addressing the molecular and functional effects of CBD in response to exercise are required to further elucidate the ergogenic potential of CBD.

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.

Transcriptome Response of Differentiating Muscle Satellite Cells to Thermal Challenge in Commercial Turkey.

Early muscle development involves the proliferation and differentiation of stem cells (satellite cells, SCs) in the mesoderm to form multinucleated myotubes that mature into muscle fibers and fiber bundles. Proliferation of SCs increases the number of cells available for muscle formation while simultaneously maintaining a population of cells for future response. Differentiation dramatically changes properties of the SCs and environmental stressors can have long lasting effects on muscle growth and physiology. This study was designed to characterize transcriptional changes induced in turkey SCs undergoing differentiation under thermal challenge. Satellite cells from the pectoralis major (p. major) muscle of 1-wk old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (Randombred Control Line 2, RBC2) were proliferated for 72 h at 38 °C and then differentiated for 48 h at 33 °C (cold), 43 °C (hot) or 38 °C (control). Gene expression among thermal treatments and between turkey lines was examined by RNAseq to detect significant differentially expressed genes (DEGs). Cold treatment resulted in significant gene expression changes in the SCs from both turkey lines, with the primary effect being down regulation of the DEGs with overrepresentation of genes involved in regulation of skeletal muscle tissue regeneration and sarcomere organization. Heat stress increased expression of genes reported to regulate myoblast differentiation and survival and to promote cell adhesion particularly in the NCT line. Results suggest that growth selection in turkeys has altered the developmental potential of SCs in commercial birds to increase hypertrophic potential of the p. major muscle and sarcomere assembly. The biology of SCs may account for the distinctly different outcomes in response to thermal challenge on breast muscle growth, development, and structure of the turkey.

RNA Sequencing Reveals the Regulation of Betaine on Chicken Myogenesis.

Simple SummaryBetaine is a healthy source of methyl and glycine and a widely used feed additive to promote animal growth. Previous studies mainly focused on its anti-osmotic pressure, anti-inflammatory, and growth-promoting effect in vitro. Still, poultry’s growth-promoting mechanism and regulation of muscle cells remain unknown. In this study, we found that a low concentration of betaine could inhibit myoblast cell proliferation but promote myotube formation, suggesting that the growth-promoting effect of betaine was achieved through the differentiation and fusion of myotubes. In addition, RNA sequencing found a series of betaine-affected genes, which could provide a theoretical basis for explaining betaine’s regulation on chicken myogenesis in vitro.Betaine is trimethylglycine and a universal methyl donor which could provide methyl and glycine for cells and animals. As a new star in epigenetics, N6-Methyladenosine has been reported to regulate multiple biological activities, but the regulatory mechanism of betaine on N6-Methyladenosine as well as myogenesis was little studied. In this study, we treated chicken primary myoblast cells with different concentrations of betaine (0, 10, 25, and 50 mmol/L) and found that myoblast cell proliferation was inhibited, although the cell cycle was promoted in the S phase by betaine, where the myotube area was increased as well as the differentiation marker genes MyoD, MyoG, MyHC, Myomarker, and Ckm. RNA sequencing obtained a total of 61 differentially expressed genes (DEGs); DEGs caused by 50 mmol/L betaine were mainly enriched in the regulation of skeletal muscle tissue regeneration and some amino acid metabolic processes. The gene expression pattern trends of all DEGs were mainly clustered into 2 profiles, with the increase in betaine concentration, the gene expression pattern either increased or decreased continuously. Overall, a low concentration betaine can increase the N6-Methyladenosine modification level and myotube area but depresses myoblast cell proliferation in vitro.

Sex-Based Differences in the Myogenic Response and Inflammatory Gene Expression Following Eccentric Contractions in Humans.

After muscle injury, the interaction between muscle satellite cells (SC) and the immune response is instrumental for the repair and regeneration of skeletal muscle tissue. Studies have reported sex-based differences in the skeletal muscle inflammatory and regenerative response following injury. However, many of these studies investigated such differences by manipulating the concentration of estradiol, in rodents and humans, without directly comparing males to females. We sought to explore differences in the myogenic and inflammatory response following unaccustomed eccentric exercise in males and females. We hypothesized that females would have a blunted myogenic and inflammatory response as compared to males. Methods: 26 (13 male, 13 female) healthy young adults (22 ± 0.4 years [mean ± SEM]) performed 300 maximal eccentric contractions (180°/s) of the knee extensors. Muscle biopsies were taken before (pre) and 48 h (post) following eccentric damage. SC content and activation were determined by immunohistochemical and real time-polymerase chain reaction (rt-PCR) analysis. Inflammatory markers were analyzed using rt-PCR. Results: Following eccentric damage, males had a greater expansion of type I-associated SC (p < 0.05), and there was a trend for a greater expansion in total SC (type I + II fibers) (p = 0.06) compared to females. There was a trend for a greater increase in Pax7 and CCL2 gene expression in males compared to females (p = 0.09). Conclusion: We conclude that there are sex-based differences in the myogenic and inflammatory response, where females have a blunted SC and inflammatory response.

Injectable laminin-biofunctionalized gellan gum hydrogels loaded with myoblasts for skeletal muscle regeneration

Moderate muscular injuries that exceed muscular tissue's auto-healing capacity are still a topic of noteworthy concern. Tissue engineering appeared as a promising therapeutic strategy capable of overcoming this unmet clinical need. To attain such goal, herein we propose an in situ-crosslinking gellan gum (GG)-based hydrogel tethered with a skeletal muscle-inspired laminin-derived peptide RKRLQVQLSIRTC(Q) and encapsulated with skeletal muscle cells (SMCs). Pre-hydrogel solutions presented decreasing shear viscosity with increasing shear rate and shear stress, and required low forces for extrusion, validating their injectability. The GGDVS hydrogel was functionalized with Q-peptide with 30% of efficiency. C2C12 were able to adhere to the developed hydrogel, remained living and spreading 7 days post-encapsulation. Q-peptide release studies indicated that 25% of the unbound peptide can be released from the hydrogels up to 7 days, dependent on the hydrogel formulation. Treatment of a chemically-induced muscular lesion in mice with an injection of C2C12-laden hydrogels improved myogenesis, primarily promoted by the C2C12. In accordance, a high density of myoblasts (α-SA+ and MYH7+) were localized in tissues treated with the C2C12 (alone or encapsulated in the hydrogel). α-SA protein levels were significantly increased 8 weeks post-treatment with C2C12-laden hydrogels and MHC protein levels were increased in all experimental groups 4 weeks post-treatment, in relation to the SHAM. Neovascularization and neoinnervation was also detected in the defects. Altogether, this study indicates that C2C12-laden hydrogels hold great potential for skeletal muscle regeneration. Statement of significanceWe developed an injectable gellan gum-based hydrogel for delivering C2C12 into localized myopathic model. The gellan gum was biofunctinalized with laminin-derived peptide to mimic the native muscular ECM. In addition, hydrogel was physically tuned to mimic the mechanical properties of native tissue. To the best of our knowledge, this formula was used for the first time under the context of skeletal muscle tissue regeneration. The injectability of the developed hydrogel provided non-invasive administration method, combined with a reliable microenvironment that can host C2C12 with nominal inflammation, indicated by the survival and adhesion of encapsulated cells post-injection. The treatment of skeletal muscle defect with the cell-laden hydrogel approach significantly enhanced the regeneration of localized muscular trauma.

Photoreactive Hydrogel Stiffness Influences Volumetric Muscle Loss Repair.

Volumetric muscle loss (VML) injuries are characterized by permanent loss of muscle mass, structure, and function. Hydrogel biomaterials provide an attractive platform for skeletal muscle tissue engineering due to the ability to easily modulate their biophysical and biochemical properties to match a range of tissue characteristics. In this work, we successfully developed a mechanically tunable hyaluronic acid (HA) hydrogel system to investigate the influence of hydrogel stiffness on VML repair. HA was functionalized with photoreactive norbornene groups to create hydrogel networks that rapidly crosslink through thiol-ene click chemistry with tailored mechanics. Mechanical properties were controlled by modulating the amount of matrix metalloproteinase-degradable peptide crosslinker to produce hydrogels with increasing elastic moduli of 1.1 ± 0.002, 3.0 ± 0.002, and 10.6 ± 0.006 kPa, mimicking a relevant range of developing and mature muscle stiffnesses. Functional muscle recovery was assessed following implantation of the HA hydrogels by in situ photopolymerization into rat latissimus dorsi (LD) VML defects at 12 and 24 weeks postinjury. After 12 weeks, muscles treated with medium stiffness (3.0 kPa) hydrogels produced maximum isometric forces most similar to contralateral healthy LD muscles. This trend persisted at 24 weeks postinjury, suggestive of sustained functional recovery. Histological analysis revealed a significantly larger zone of regeneration with more de novo muscle fibers following implantation of medium stiffness hydrogels in VML-injured muscles compared to other experimental groups. Lower (low and medium) stiffness hydrogels also appeared to attenuate the chronic inflammatory response characteristic of VML injuries, displaying similar levels of macrophage infiltration and polarization to healthy muscle. Together these findings illustrate the importance of hydrogel mechanical properties in supporting functional repair of VML injuries. Impact statement This report defines the role hydrogel mechanical properties play in the repair of volumetric muscle loss (VML) injuries. We show that an intermediate hydrogel stiffness (3 kPa) more compliant than adult muscle tissue facilitated improved and sustained regenerative outcomes up to 24 weeks postinjury in a rat latissimus dorsi model of VML. Muscles treated with 3 kPa hydrogels showed enhanced myogenesis and attenuation of the chronic inflammatory response characteristic of VML injuries. These results should help guide the future design of hydrogels for skeletal muscle tissue engineering and regeneration.

3D Electrospun Nanofiber-Based Scaffolds: From Preparations and Properties to Tissue Regeneration Applications.

Electrospun nanofibers have been frequently used for tissue engineering due to their morphological similarities with the extracellular matrix (ECM) and tunable chemical and physical properties for regulating cell behaviors and functions. However, most of the existing electrospun nanofibers have a closely packed two-dimensional (2D) membrane with the intrinsic shortcomings of limited cellular infiltration, restricted nutrition diffusion, and unsatisfied thickness. Three-dimensional (3D) electrospun nanofiber-based scaffolds can provide stem cells with 3D microenvironments and biomimetic fibrous structures. Thus, they have been demonstrated to be good candidates for in vivo repair of different tissues. This review summarizes the recent developments in 3D electrospun nanofiber-based scaffolds (ENF-S) for tissue engineering. Three types of 3D ENF-S fabricated using different approaches classified into electrospun nanofiber 3D scaffolds, electrospun nanofiber/hydrogel composite 3D scaffolds, and electrospun nanofiber/porous matrix composite 3D scaffolds are discussed. New functions for these 3D ENF-S and properties, such as facilitated cell infiltration, 3D fibrous architecture, enhanced mechanical properties, and tunable degradability, meeting the requirements of tissue engineering scaffolds were discovered. The applications of 3D ENF-S in cartilage, bone, tendon, ligament, skeletal muscle, nerve, and cardiac tissue regeneration are then presented with a discussion of current challenges and future directions. Finally, we give summaries and future perspectives of 3D ENF-S in tissue engineering and clinical transformation.

Current Strategies for the Regeneration of Skeletal Muscle Tissue.

Traumatic injuries, tumor resections, and degenerative diseases can damage skeletal muscle and lead to functional impairment and severe disability. Skeletal muscle regeneration is a complex process that depends on various cell types, signaling molecules, architectural cues, and physicochemical properties to be successful. To promote muscle repair and regeneration, various strategies for skeletal muscle tissue engineering have been developed in the last decades. However, there is still a high demand for the development of new methods and materials that promote skeletal muscle repair and functional regeneration to bring approaches closer to therapies in the clinic that structurally and functionally repair muscle. The combination of stem cells, biomaterials, and biomolecules is used to induce skeletal muscle regeneration. In this review, we provide an overview of different cell types used to treat skeletal muscle injury, highlight current strategies in biomaterial-based approaches, the importance of topography for the successful creation of functional striated muscle fibers, and discuss novel methods for muscle regeneration and challenges for their future clinical implementation.

Inhibiting PHD2 in human periodontal ligament cells via lentiviral vector-mediated RNA interference facilitates cell osteogenic differentiation and periodontal repair.

Periodontal defect regeneration in severe periodontitis remains a challenging task in clinic owing to poor survival of seed cells caused by the remaining oxidative stress microenvironment. Recently, the reduction of prolyl hydroxylase domain-containing protein 2 (PHD2), a primary cellular oxygen sensor, has shown an incredible extensive effect on skeletal muscle tissue regeneration by improving cell resistance to reactive oxygen species, whereas its role in periodontal defect repair is unclear. Here, through lentivirus vector-mediated RNA interference, the PHD2 gene in human periodontal ligament cells (hPDLCs) is silenced, leading to hypoxia-inducible factor-1α stabilization in normoxia. In vitro, PHD2 silencing not only exhibited a satisfactory effect on cell proliferation, but also induced distinguished osteogenic differentiation of hPDLCs. Real-time polymerase chain reaction and Western blotting revealed significant up-regulation of osteocalcin, alkaline phosphatase (ALP), runt-related transcription factor 2, and collagen type I (COL I). Under oxidative stress conditions, COL I and ALP expression levels, suppressed by 100 μM H2 O2 , were elevated by PHD2-gene-silencing in hPDLCs. In vivo, periodontal fenestration defects were established in 18 female Sprague-Dawley rats aged 6wk old, followed by implantation of PHD2 silencing hPDLCs in situ for 21 d. Persistent and stable silencing of PHD2 in hPDLCs promoted better new bone formation according to microcomputed tomography 3D reconstruction and related bone parameter analysis. This work demonstrates the therapeutic efficiency of PHD2 gene interference in osteogenic differentiation and periodontal defect repair for highly efficient periodontal regeneration.

Applications of Small Molecules in Muscle Tissue Engineering

Introduction: Skeletal muscles account for about 40% of the total body weight. Every year, hundreds of people lose at least part of their muscle tissue due to illness, war, and accidents. This can lead to disruption of activities such as breathing, movement, and social life. To this end, various therapeutic strategies such as medication therapy, cell therapy and tissue transplantation have been used or studied in muscle regeneration. However, there is no effective and well-defined clinical approach for treatment of muscle injuries and the severity of muscle injuries increase with age in most cases. Therefore, investigation for finding new and effective clinical approach for muscle regeneration is one of the most important issues in basic and clinical researches. Tissue engineering is considered as one of the promising and newest approach for skeletal muscle tissue regeneration and provides an appropriate model for personalized medicine and basic researches that can be used in personalized medicine and basic research. Besides biomaterials and cells, inducing factors are another element of tissue engineering. These factors influence epigenetic mechanisms and signaling pathway, thereby inducing proliferation, differentiation, and migration of cells used in muscle tissue engineering, and accelerates muscle formation in vitro. Recently, small molecules have been used as alternatives to growth factors or along with other inducing factors in muscle tissue engineering. Since they do not induce an immune reaction, penetrate easily to the cells and have a specific molecular target, therefore they have attracted much attention as the cost-effective inducing factors in tissue engineering. Conclusion: Taken together, the effective small molecules in muscle tissue engineering can be used with different biomaterial conditions (e.g. hydrogel, decellularized tissue, and synthetic scaffolds) in both in vivo and in vitro, resulting to production of cost effective and highly efficient engineered muscle tissues that help to achieve therapeutical goals of muscle tissue engineering. Herein, we describe tissue engineering and review the small molecules used in skeletal muscle tissue engineering.