Response Of Myoblasts Research Articles

Process-Dependent Variations in the Proliferation of Myoblasts, Fibroblasts and Chondrocytes on Laser-Sintered Polypropylene.

Additively manufactured polyolefins find broad applications in medical engineering, enabling the manufacturing of patient-specific geometries. For investigating the influence of processing conditions of laser sintered locally macroporous polypropylene substrates, the response of myoblasts, chondrocytes, and fibroblasts has been characterized in this study. An influence of the applied manufacturing parameters on the attachment and viability of the investigated cells is observed, showing the effect of the superficial pore topology on the attachment and the spreading of cells. The viability and attachment of fibroblasts and chondrocytes could be improved by reducing the thermal exposure during the processing step of the dense base part, associated with increased superficial porosity and the corresponding increase of the surface area. The applied additive manufacturing process of macroporous structures influences emerging cell morphologies, leading to an extended morphological expression of chondrocytes and the overgrowth of small pores by fibroblasts. This indicates an improvement in superficial cell adhesion due to larger pores. These findings indicate the significance of the processing conditions in laser sintering of polypropylene on the cell response through the optimization of processing parameters and the attachment of an open-cell pore structure.

Influence of metal ions on the isothermal self-assembly of DNA nanostructures.

DNA nanostructures are typically assembled by thermal annealing in buffers containing magnesium. We demonstrate the assembly of DNA nanostructures at constant temperatures ranging from 4 °C to 50 °C in solutions containing different metal ions. The choice of metal ions and the assembly temperature influence the isothermal assembly of several DNA motifs and designed three-dimensional DNA crystals. Molecular dynamics simulations show more fluctuations of the DNA structure in select monovalent ions (Na+ and K+) compared to divalent ions (Mg2+ and Ca2+). A key highlight is the successful assembly of DNA motifs in nickel-containing buffer at temperatures below 40 °C, otherwise unachievable at higher temperatures, or using an annealing protocol. DNA nanostructures isothermally assembled in different ions do not affect the viability of fibroblasts, myoblasts, and myotubes and or the immune response in myoblasts. The use of ions other than the typically-used magnesium holds key potential in biological and materials science applications that require minimal amounts of magnesium.

Proteome Alterations and Nucleosome Activation in Rat Myoblasts Treated with Cerium Oxide Nanoparticles.

Oxidative stress is a widespread causative agent of disease. Together with its general relevance for biomedicine, such a dynamic is recognizably detrimental to space exploration. Among other solutions, cerium oxide nanoparticles (or nanoceria, NC) display a long-lasting, self-renewable antioxidant activity. In a previous experiment, we evaluated oxidative imbalance in rat myoblasts in space, aboard the International Space Station, and unveiled possible protective effects from NC through RNA sequencing. Here, we focus on the myoblast response to NC on land by means of proteomics, defining a list of proteins that putatively react to NC and confirming nucleosomes/histones as likely mediators of its molecular action. The proteomics data set we present here and its counterpart from the space study share four factors. These are coherently either up- (Hist1h4b) or down-regulated (Gnl3, Mtdh, Trip12) upon NC exposure.

Matrix Nonlinear Viscoelasticity Regulates Skeletal Myogenesis through MRTF Nuclear Localization and Nuclear Mechanotransduction.

Mechanically sensitive tissues (e.g., skeletal muscles) greatly need mechanical stimuli during the development and maturation. The extracellular matrix (ECM) mediates these signals through nonlinear viscoelasticity of collagen networks that are predominant components of the ECM. However, the interactions between cells and ECM form a feedback loop, and it has not yet been possible to determine the degree to which, if any, of the features of matrix nonlinear viscoelasticity affect skeletal muscle development and regeneration. In this study, a nonlinear viscoelastic feature (i.e., strain-enhanced stress relaxation (SESR)) in normal skeletal muscles is observed, which however is almost absent in diseased muscles from Duchenne muscular dystrophy mice. It is recapitulated such SESR feature in vitro and separated the effects of mechanical strain and ECM viscoelasticity on myoblast response by developing a collagen-based hydrogel platform. Both strain and stress relaxation induce myogenic differentiation and myotube formation by C2C12 myoblasts, and myogenesis is more promoted by applying SESR. This promotion can be explained by the effects of SESR on actin polymerization-mediated myocardin related transcription factor (MRTF) nuclear localization and nuclear mechanotransduction. This study represents the first attempt to investigate the SESR phenomenon in skeletal muscles and reveal underlying mechanobiology, which will provide new opportunities for the tissue injury treatments.

Secretome from myoblasts statically loaded at low intensity promotes tenocyte proliferation via the IGF-1 receptor pathway.

Exercise is widely recognized as beneficial for tendon healing. Recently, it has been described that muscle-derived molecules secreted in response to static exercise influence tendon healing. In this study, the optimal static loading intensity for tendon healing and the composition of secretome released by myoblasts in response to different intensities of static strain were investigated. In an in vitro coculture model, myoblasts were mechanically loaded using a Flexcell Tension System. Tenocytes were seeded on transwell inserts that allowed communication between the tenocytes and myoblasts without direct contact. Proliferation and migration assays, together with RNA sequencing, were used to determine potential cellular signaling pathways. The secretome from myoblasts exposed to 2% static loading increased the proliferation and migration of the cocultured tenocytes. RNA-seq analysis revealed that this loading condition upregulated the expression of numerous genes encoding secretory proteins, including insulin-like growth factor-1 (IGF-1). Confirmation of IGF-1 expression and secretion was carried out using qPCR and enzyme-linked immunosorbt assay (ELISA), revealing a statistically significant upregulation in response to 2% static loading in comparison to both control conditions and higher loading intensities of 5% and 10%. Addition of an inhibitor of the IGF-1 receptor (PQ401) to the tenocytes significantly reduced myoblast secretome-induced tenocyte proliferation. In conclusion, IGF-1 may be an important molecule in the statically loaded myoblast secretome, which is responsible for influencing tenocytes during exercise-induced healing.

Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in C2C12 myoblasts by modulating the β-catenin/NF-κB signaling pathway

A myogenetic oligodeoxynucleotide, iSN04, is the 18-base single-stranded DNA that acts as an anti-nucleolin aptamer. iSN04 has been reported to restore myogenic differentiation by suppressing inflammatory responses in myoblasts isolated from patients with diabetes or healthy myoblasts exposed to cancer-releasing factors. Thus, iSN04 is expected to be a nucleic acid drug for the muscle wasting associated with chronic diseases. The present study investigated the anti-inflammatory mechanism of iSN04 in the murine myoblast cell line C2C12. Tumor necrosis factor-α (TNF-α) or Toll-like receptor (TLR) ligands (Pam3CSK4 and FSL-1) induced nuclear translocation and transcriptional activity of nuclear factor-κB (NF-κB), resulting in upregulated expression of TNF-α and interleukin-6. Pre-treatment with iSN04 significantly suppressed these inflammatory responses by inhibiting the nuclear accumulation of β-catenin induced by TNF-α or TLR ligands. These results demonstrate that antagonizing nucleolin with iSN04 downregulates the inflammatory effect mediated by the β-catenin/NF-κB signaling pathway in C2C12cells. In addition, the anti-inflammatory effects of iSN04 were also observed in the rat smooth muscle cell line A10 and the murine adipocyte-like fibroblast cell line 3T3-L1, suggesting that iSN04 may be useful in preventing inflammation induced by metabolic disorders.

Hippo pathway inhibition promotes metabolic adaptability and antioxidant response in myoblasts

Metabolic plasticity in a hostile environment ensures cell survival. We investigated whether Hippo pathway inhibition contributed to cell adaptations under challenging conditions. We examined metabolic profiles and fuel substrate choices and preferences in C2C12 myoblasts after Hippo pathway inhibition via Salvador knockdown (SAV1 KD). SAV1 KD induced higher ATP production and a more energetic phenotype. Bioenergetic profiling showed enhanced key mitochondrial parameters including spare respiratory capacity. SAV1 KD cells showed markedly elevated glycolysis and glycolytic reserves; blocking other fuel-oxidation pathways enhanced mitochondrial flexibility of glucose oxidation. Under limited glucose, endogenous fatty acid oxidation increased to cope with bioenergetic stress. Gene expression patterns after SAV1 KD suggested transcriptional upregulation of key metabolic network regulators to promote energy production and free radical scavenging that may prevent impaired lipid and glucose metabolism. In SAV1 KD cells, sirtuin signaling was the top enriched canonical pathway linked with enhanced mitochondrial ATP production. Collectively, we demonstrated that Hippo pathway inhibition in SAV1 KD cells induces multiple metabolic properties, including enhancing mitochondrial spare respiratory capacity or glycolytic reserve to cope with stress and upregulating metabolic pathways supporting elevated ATP demand, bioenergetics, and glycolysis and counteracting oxidative stress. In response to metabolic challenges, SAV1 KD cells can increase fatty acid oxidation or glucose-coupled oxidative phosphorylation capacity to compensate for substrate limitations or alternative fuel oxidation pathway inhibition.

HDAC8 regulates protein kinase D phosphorylation in skeletal myoblasts in response to stress signaling

Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination of myogenic transcription factors. Myogenic transcription is tightly regulated by various intracellular signaling pathways, which include members of the protein kinase D (PKD) family. PKD is a family of serine-threonine kinases that regulate gene expression, protein secretion, cell proliferation, differentiation and inflammation. PKD is a unique PKC family member that shares distant sequence homology to calcium-regulated kinases and plays an important role in muscle physiology. In this report, we show that class I histone deacetylase (HDAC) inhibition, and in particular HDAC8 inhibition, attenuated PKD phosphorylation in skeletal C2C12 myoblasts in response to phorbol ester, angiotensin II and dexamethasone signaling independent of changes in total PKD protein expression. As class I HDACs and PKD signaling are requisite for myocyte differentiation, these data suggest that HDAC8 functions as a potential feedback regulator of PKD phosphorylation to control myogenic gene expression.

Ablation of PRAK exacerbates the mitochondria dysfunction in C2C12 myoblasts exposed to palmitate stress

Backgroundp38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating survival and metabolism. However, the role of PRAK in the regulation of palmitate‐induced metabolic stress remains unknown. We examined whether deletion of PRAK would suppress cell survival and mitochondrial function and whether PRAK‐induced detrimental effect was mitigated by irisin in C2C12 myoblasts in response to palmitate (PA) stress.MethodsPRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas‐9 genome editing technology. Both wild type and PRAK‐/‐ C2C12 cells were exposed to palmitate at the concentration of 200 mmol/L for 48 h to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK‐/‐cells was determined to explore its relationship with RPAK in response to palmitate stress. Cell viability, ATP product, glucose uptake, mitochondrial membrane potential and mitochondrial swelling were assessed.ResultsPRAK knockout decreased C2C12 viability in response to PA stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to PA treatment, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential and increasing mitochondrial swelling. However, PRAK knockout induced an impairment of mitochondrial and cell damage in C2C12 cells exposed to PA stress were rescued by treatment of irisin (10ng/ml).ConclusionOur finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function and glucose uptake in C2C12 cells exposed to palmitate induced metabolic stress.

Inflammatory Caspase Activity Mediates HMGB1 Release and Differentiation in Myoblasts Affected by Peripheral Arterial Disease.

Introduction: We previously showed that caspase-1 and -11, which are activated by inflammasomes, mediate recovery from muscle ischemia in mice. We hypothesized that similar to murine models, inflammatory caspases modulate myogenicity and inflammation in ischemic muscle disease. Methods: Caspase activity was measured in ischemic and perfused human myoblasts in response to the NLRP3 and AIM2 inflammasome agonists (nigericin and poly(dA:dT), respectively) with and without specific caspase-1 or pan-caspase inhibition. mRNA levels of myogenic markers and caspase-1 were assessed, and protein levels of caspases-1, -4, -5, and -3 were measured by Western blot. Results: When compared to perfused cells, ischemic myoblasts demonstrated attenuated MyoD and myogenin and elevated caspase-1 mRNA. Ischemic myoblasts also had significantly higher enzymatic caspase activity with poly(dA:dT) (p < 0.001), but not nigericin stimulation. Inhibition of caspase activity including caspase-4/-5, but not caspase-1, blocked activation effects of poly(dA:dT). Ischemic myoblasts had elevated cleaved caspase-5. Inhibition of caspase activity deterred differentiation in ischemic but not perfused myoblasts and reduced the release of HMGB1 from both groups. Conclusion: Inflammatory caspases can be activated in ischemic myoblasts by AIM2 and influence ischemic myoblast differentiation and release of pro-angiogenic HMGB1. AIM2 inflammasome involvement suggests a role as a DNA damage sensor, and our data suggest that caspase-5 rather than caspase-1 may mediate the downstream mediator of this pathway.

Hybrid Self‐Assembling Peptide/Gelatin Methacrylate (GelMA) Bioink Blend for Improved Bioprintability and Primary Myoblast Response

Organ fabrication as the solution to renewable donor demands requires the ability to spatially deposit viable cells into biologically relevant constructs; necessitating reliable and effective cell deposition through bioprinting and the subsequent ability to mature. However, effective bioink development demands advances in both printability and control of cellular response. Effective bioinks are designed to retain shape fidelity, influence cellular behavior, having bioactive morphologies stiffness and highly hydrated environment. Hybrid hydrogels are promising candidates as they reduce the need to re‐engineer materials for tissue‐specific properties, with each component offering beneficial properties. Herein, a multicomponent bioink is developed whereby gelatin methacrylate (GelMA) and fluorenylmethoxycarbonyprotected self‐assembling peptides (Fmoc‐SAPs) undergo coassembly to yield a tuneable bioink. This study shows that the reported fibronectin‐inspired fmoc‐SAPs present cell attachment epitopes RGD and PHSRN in the form of bioactive nanofibers; and that the GelMA enables superior printability, stability in media, and controlled mechanical properties. Importantly, when in the hybrid format, no disruption to either the methacrylate crosslinking of GelMA, or self‐assembled peptide fibril formation is observed. Finally, studies with primary myoblasts show over 98% viability at 72 h and differentiation into fused myotubes at one and two weeks demonstrate the utility of the material as a functional bioink for muscle engineering.

Deletion of PRAK Mitigates the Mitochondria Function and Suppresses Insulin Signaling in C2C12 Myoblasts Exposed to High Glucose.

Background: p38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating cell death and survival. However, the role of PRAK in the regulation of metabolic stress remains unclear. We examined the effects of PRAK on cell survival and mitochondrial function in C2C12 myoblasts in response to high glucose stresses. Methods: PRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas-9 genome editing technology. Both wild type and PRAK−/− C2C12 cells were exposed to high glucose at the concentration of 30 mmol/L to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK−/− cells was determined to explore its relationship with RPAK. Cell viability, ATP product, glucose uptake, mitochondrial damage, and insulin signaling were assessed. Results: PRAK knockout decreased C2C12 viability in response to high glucose stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to high glucose, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential. PRAK knockout induced impairment of mitochondrial and cell damage were rescued by irisin. PRAK knockout caused decrease in phosphorylated PI3 kinase at Tyr 485, IRS-1 and AMPKα and but did not affect non-phosphorylated PI3 kinase, IRS-1 and AMPKα signaling. High glucose caused the further reduction of phosphorylated PI3 kinase, IRS-1 and AMPKα. Irisin treatment preserved phosphorylated PI3 kinase, IRS-1by rescuing PRAK in high glucose treatment. Conclusion: Our finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function, and high glucose stress.

Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines

Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts.

ABSTRACTImbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionarily conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing ER structures. We demonstrate that the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process that does not depend on autophagosome–lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.This article has an associated First Person interview with the first author of the paper.

FSHD / OPMD / MYOTONIC DYSTROPHY: P.240 Targeting unfolded protein response to resolve aggresomes accumulation in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare late-onset genetic myopathy, affecting mainly eyelid and pharyngeal muscles leading to ptosis and dysphagia, respectively. OPMD is caused by a mutation in Poly-A Binding Protein Nuclear 1 (PABPN1) gene, that codes for a protein essential for proper mRNA biogenesis in nucleus. Accordingly, mutated PABPN1 in OPMD results in PABPN1 intranuclear aggregates in muscle that sequestrate mRNA and proteins and thus induce a wide range of deleterious consequences on muscle homeostasis (mitochondrial dysfunctions, calcium homeostasis disruption, fibrosis, force impairment, atrophy). We previously demonstrated that mouse model of OPMD presents muscle Endoplasmic Reticulum (ER) stress, i.e. an accumulation of misfolded proteins in ER lumen, and that restoring the physiological and resolutive Unfolded Protein Response (UPR) through guanabenz acetate treatment is efficient to decrease PABPN1 nuclear aggregates and improve muscle function in OPMD mouse (Malerba et al Hum Mol Genet 2019). To validate these results in human, we now analyze muscle biopsies from OPMD patients compared to healthy donors and observe a disruption of both key genes and proteins of UPR signalling in OPMD muscles. Interestingly, we find that the alteration is positively correlated with the clinical state of the muscle. Furthermore, we detect GRP78 (BiP), the most important ER chaperone, trapped in PABPN1 nuclear aggregates, suggesting a reduced folding capacity in ER. As a consequence, OPMD muscles present an accumulation of large beta-sheet protein aggregates called aggresomes. Consistently, we observe that human OPMD myoblasts are more sensible to develop aggresomes than healthy myoblasts in response to ER stress. Overall, our results unravel an impairment of ER folding and UPR as well as an aggresomes accumulation in human OPMD muscle. Thus, restoring the protein folding in ER as well as improving the aggresomes clearance could be a promising therapeutic strategy against OPMD.

Effects of a human microenvironment on the differentiation of human myoblasts

Myogenic differentiation mechanisms are generally assessed using a murine cell line placed in low concentrations of an animal-derived serum. To more closely approximate invivo pathophysiological conditions, recent studies have combined the use of human muscle cells with human serum. Nevertheless, the invitro studies of the effects of a human microenvironment on the differentiation process of human myoblasts require the identification of the culture conditions that would provide an optimal and reproducible differentiation process of human muscle cells. We assessed the differentiation variability resulting from the use of human myoblasts and serums from healthy subjects by measuring the myotube diameter, fusion index and surface covered by myotubes. We showed the preserved cell-dependent variability of the differentiation response of myoblasts cultured in human serums compared to FBS. We found that using a pool of serums reduced the serum-dependent variability of the myogenic response compared to individual serums. We validated our methodology by showing the atrophying effect of pooled serums from COPD patients on healthy human myotubes. By replacing animal-derived tissues with human myoblasts and serums, and by validating the sensitivity of cultured human muscle cells to a pathological microenvironment, this human cell culture model offers a valuable tool for studying the role of the microenvironment in chronic disease.

Biomechanical effects on microRNA expression in skeletal muscle differentiation

We examined whether mechanical stretch affected expression of muscle-specific microRNAs (miRNAs) that regulate proliferation (miR-133a) or differentiation (miR-1, -206). Real-time quantitative RT-PCR was used to assess miRNA regulation in the murine myoblast cell line C2C12 exposed to stretch regimens that promote either proliferation (high stretch: 17%, 1 Hz) or differentiation (moderate stretch: 10%, 0.5 Hz) after adding media that promotes differentiation. Controls consisted of myoblasts cultured under static conditions. While miRNA expression was not affected by high stretch, a significant effect of stretch (P

A 3D printing strategy for fabricating in situ topographical scaffolds using pluronic F-127

Abstract Topographical cues are one of the prerequisites for successful regeneration of muscle tissue. However, fabrication methods using three-dimensional (3D) bioprinters are limited by the simple nozzle-based extrusion or uncontrollability of photo-reactive systems. Hence, most studies on inducing topographical cues were focused on two-dimensional (2D) surface structures and based on imprinting and soft-lithography processes. Although 2D patterned surfaces provide outstanding insight into optimal patterned architectures by facilitating the analysis of various myoblast responses, it can be difficult to achieve complex 3D structures with microscale topographical cues. For this reason, we propose a new strategy for obtaining topographical cues in 3D printed synthetic biopolymers for regenerating muscle tissue. A uniaxially aligned pattern was obtained on the struts of the matrix composed of poly(e-caprolactone) (PCL) or poly(lactic-co-glycolic acid) (PLGA), by taking advantage of the immiscible rheological properties and flow-induced force in the dispersed pluronic F-127 phase (sacrificial material) and matrix materials. To observe the effects of the topographical cues of the fabricated structures on cellular responses, including myogenic gene expression, myoblasts (C2C12 cells) were cultured for various lengths of time, and the micropatterned structure provided a rapid myotube formation, compared to the control, which did not undergo the microscale patterning process. In addition, to extend the printing technique, the alignment of primary tenocytes cultured on the uniaxial patterned structure was evaluated.

Micropatterned conductive hydrogels as multifunctional muscle-mimicking biomaterials: Graphene-incorporated hydrogels directly patterned with femtosecond laser ablation

Multifunctional biomaterials that can provide physical, electrical, and structural cues to cells and tissues are highly desirable to mimic the important characteristics of native tissues and efficiently modulate cellular behaviors. Especially, electrically conductive biomaterials can efficiently deliver electrical signals to living systems; however, the production of conductive biomaterials presenting multiple cell interactive cues is still a great challenge. In this study, we fabricafed an electrically conductive, mechanically soft, and topographically active hydrogel by micropatterning a graphene oxide (GO)-incorporated polyacrylamide hydrogel (GO/PAAm) with femtosecond laser ablation (FLA) and subsequent chemical reduction. FLA parameters were optimized to efficiently produce distinct line patterns on GO/PAAm hydrogels to induce myoblast alignment and maturation. The line patterns distances (PD) were varied to have different topographies (20-80 μm PD). In vitro studies with C2C12 myoblasts revealed that the micopatterned hydrogels are superior to the unpatterned substrates in inducing myogenesis and myotube alignment. Reduced GO/PAAm with 50 μm PD, i.e., PD50/r(GO/PAAm), showed the best results among the various features for differentiation and myotube alignment. Electrical stimulation of myoblasts on the micropatterned conductive hydrogels further promoted the differentiation of myoblasts. In vivo implantation studies indicated good tissue compatibility of PD50/r(GO/PAAm) samples. Altogether, we successfully demonstrated that the micropatterned r(GO/PAAm) may offer multiple properties capable of positively affecting myoblast responses. This hydrogel may serve as an effective multifunctional biomaterial, which possesses the topography for cell alignment/maturation, mechanical properties of the native skeletal muscle tissue, and desirable electrical conductivity for delivering electrical signals to cells, for various biomedical applications such as muscle tissue scaffolds. STATEMENT OF SIGNIFICANCE: Micropatterned conductive hydrogels were created by polymerization of a graphene oxide-incorporated polyacrylamide hydrogel, micropatterning with femtosecond laser ablation, and chemical reduction, which can mimic important characteristics of native skeletal muscle tissues. The micropatterned conductive hydro-gels promoted myogenesis/alignment, enabled electrical stimulation of myoblasts, and displayed good tissue compatibility, which can therefore serve as a multifunctional biomaterial that is topographically active, mechanically soft, and electrically conductive for delivering multiple cell stimulating signals for potential skeletal muscle tissue engineering applications.