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James J Hickman - One of the best experts on this subject based on the ideXlab platform.
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developing a novel serum free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in Myotube formation
In Vitro Cellular & Developmental Biology – Animal, 2009Co-Authors: James W. Rumsey, Neelima Bhargava, Lisa Riedel, Jung Fong Kang, James J Hickman, Cassie GregoryAbstract:This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate Myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a nonbiological cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine. Each growth factor in the medium was experimentally evaluated for its effect on Myotube formation. The resulting Myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust Myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle–nerve coculture. Immunocytochemical evidence of nerve–muscle coculture has also been documented. Applications for this novel culture system include biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders, and skeletal muscle tissue engineering.
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Tissue engineering intrafusal fibers: Dose- and time-dependent differentiation of nuclear bag fibers in a defined in vitro system using neuregulin 1-β-1
Biomaterials, 2008Co-Authors: John W Rumsey, Jung Fong Kang, Robert Wagner, Peter Molnar, James J HickmanAbstract:While much is known about muscle spindle structure, innervation and function, relatively few factors have been identified that regulate intrafusal fiber differentiation and spindle development. Identification of these factors will be a crucial step in tissue engineering functional muscle systems. In this study, we investigated the role of the growth factor, neuregulin 1-β-1 (Nrg 1-β-1) EGF, for its ability to influence Myotube fate specification in a defined culture system utilizing the non-biological substrate N-1[3-(trimethoxysilyl)propyl]-diethylenetriamine (DETA). Based on morphological and immunocytochemical criteria, Nrg 1-β-1 treatment of developing Myotubes increases the ratio of nuclear bag fibers to total Myotubes from 0.019 to 0.100, approximately a five-fold increase. The Myotube cultures were evaluated for expression of the intrafusal fiber-specific alpha cardiac-like myosin heavy chain and for the expression of the non-specific slow myosin heavy chain. Additionally, the expression of ErbB2 receptors on all Myotubes was observed, while phosphorylated ErbB2 receptors were only observed in Nrg 1-β-1-treated intrafusal fibers. After Nrg 1-β-1 treatment, we were able to observe the expression of the intrafusal fiber-specific transcription factor Egr3 only in fibers exhibiting the nuclear bag phenotype. Finally, nuclear bag fibers were characterized electrophysiologically for the first time in vitro. This data shows conclusively, in a serum-free system, that Nrg 1-β-1 is necessary to drive specification of forming Myotubes to the nuclear bag phenotype.
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a defined system to allow skeletal muscle differentiation and subsequent integration with silicon microstructures
Biomaterials, 2006Co-Authors: Cassie Gregory, Lisa Riedel, James J Hickman, Peter Molnar, Kerry WilsonAbstract:This work documents the development of an in vitro cell culture model consisting of a novel serum-free medium and a non-biological growth substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), to enable functional Myotube integration with cantilevers fabricated using MEMS technology. This newly developed, defined in vitro model was used to study the differentiation of fetal rat skeletal muscle and it promoted the formation of Myotubes from the dissociated rat fetal muscle cells. The Myotubes were characterized by morphological analysis, immunocytochemistry and electrophysiology. Further, it was demonstrated that when the dissociated muscle cells were plated on fabricated microcantilevers, the muscle cells aligned along the major axis of the cantilever and formed robust Myotubes. This novel system could not only find applications in skeletal muscle differentiation and biocompatibility studies but also in bioartificial muscle engineering, hybrid actuation system development, biorobotics and for a better understanding of myopathies and neuromuscular disorders.
Peter Molnar - One of the best experts on this subject based on the ideXlab platform.
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Tissue engineering intrafusal fibers: dose and time dependent differentiation of nuclear bag fibers in a defined in vitro system using Neuregulin 1-β-1
2015Co-Authors: John W Rumsey, Jung Fong Kang, Mainak Das, Robert Wagner, Peter Molnar, James JAbstract:While much is known about muscle spindle structure, innervation and function, relatively few factors have been identified that regulate intrafusal fiber differentiation and spindle development. Identification of these factors will be a crucial step in tissue engineering functional muscle systems. In this study, we investigated the role of the growth factor, neuregulin 1-β-1 EGF (Nrg1-β-1), for its ability to influence Myotube fate specification in a defined culture system utilizing the non-biological substrate DETA. Based on morphological and immunocytochemical criteria, Nrg 1-β-1 treatment of developing Myotubes increases the ratio of nuclear bag fibers to total Myotubes from 0.019 to 0.100, approximately a five-fold increase. The Myotube cultures were evaluated for expression of the intrafusal fiber specific alpha cardiac-like myosin heavy chain and for the expression of the nonspecific slow myosin heavy chain. Additionally, the expression of ErbB2 receptors on all Myotubes was observed, while phosphorylated ErbB2 receptors were only observed in Nrg1-β-1 treated intrafusal fibers. After Nrg1-β-1 treatment, we were able to observe the expression of the intrafusal fiber specific transcription factor Egr3 only in fibers exhibiting the nuclear bag phenotype. Finally, nuclear bag fibers were characterized electrophysiologically for the first time in vitro. This data shows conclusively, in a serum-free system, that Nrg 1-β-1 is necessary to drive specification of forming Myotubes to the nuclear bag phenotype
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Tissue engineering intrafusal fibers: Dose- and time-dependent differentiation of nuclear bag fibers in a defined in vitro system using neuregulin 1-β-1
Biomaterials, 2008Co-Authors: John W Rumsey, Jung Fong Kang, Robert Wagner, Peter Molnar, James J HickmanAbstract:While much is known about muscle spindle structure, innervation and function, relatively few factors have been identified that regulate intrafusal fiber differentiation and spindle development. Identification of these factors will be a crucial step in tissue engineering functional muscle systems. In this study, we investigated the role of the growth factor, neuregulin 1-β-1 (Nrg 1-β-1) EGF, for its ability to influence Myotube fate specification in a defined culture system utilizing the non-biological substrate N-1[3-(trimethoxysilyl)propyl]-diethylenetriamine (DETA). Based on morphological and immunocytochemical criteria, Nrg 1-β-1 treatment of developing Myotubes increases the ratio of nuclear bag fibers to total Myotubes from 0.019 to 0.100, approximately a five-fold increase. The Myotube cultures were evaluated for expression of the intrafusal fiber-specific alpha cardiac-like myosin heavy chain and for the expression of the non-specific slow myosin heavy chain. Additionally, the expression of ErbB2 receptors on all Myotubes was observed, while phosphorylated ErbB2 receptors were only observed in Nrg 1-β-1-treated intrafusal fibers. After Nrg 1-β-1 treatment, we were able to observe the expression of the intrafusal fiber-specific transcription factor Egr3 only in fibers exhibiting the nuclear bag phenotype. Finally, nuclear bag fibers were characterized electrophysiologically for the first time in vitro. This data shows conclusively, in a serum-free system, that Nrg 1-β-1 is necessary to drive specification of forming Myotubes to the nuclear bag phenotype.
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a defined system to allow skeletal muscle differentiation and subsequent integration with silicon microstructures
Biomaterials, 2006Co-Authors: Cassie Gregory, Lisa Riedel, James J Hickman, Peter Molnar, Kerry WilsonAbstract:This work documents the development of an in vitro cell culture model consisting of a novel serum-free medium and a non-biological growth substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA), to enable functional Myotube integration with cantilevers fabricated using MEMS technology. This newly developed, defined in vitro model was used to study the differentiation of fetal rat skeletal muscle and it promoted the formation of Myotubes from the dissociated rat fetal muscle cells. The Myotubes were characterized by morphological analysis, immunocytochemistry and electrophysiology. Further, it was demonstrated that when the dissociated muscle cells were plated on fabricated microcantilevers, the muscle cells aligned along the major axis of the cantilever and formed robust Myotubes. This novel system could not only find applications in skeletal muscle differentiation and biocompatibility studies but also in bioartificial muscle engineering, hybrid actuation system development, biorobotics and for a better understanding of myopathies and neuromuscular disorders.
Serge Ostrovidov - One of the best experts on this subject based on the ideXlab platform.
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Enhanced skeletal muscle formation on microfluidic spun gelatin methacryloyl (GelMA) fibres using surface patterning and agrin treatment.
Journal of tissue engineering and regenerative medicine, 2018Co-Authors: Majid Ebrahimi, Serge Ostrovidov, Hojae Bae, Sahar Salehi, Sang Bok Kim, Ali KhademhosseiniAbstract:Bioengineered functional muscle tissues are beneficial for regenerative medicine due to their treatment potential for various debilitating disorders, including myopathy and traumatic injuries. However, the contractile properties of engineered muscle constructs are lacking compared with their native counterparts. Here, we used microfluidic spinning to fabricate photocrosslinkable gelatin methacryloyl (GelMA) hydrogel fibres with well-defined surface morphologies for engineering muscle tissues. We examined whether the combination of topographical cues from surface micropatterning and biochemical stimulation with recombinant agrin can improve the generation of bioengineered muscle tissue. Topographical cues on micropatterned fibres promoted alignment of C2C12 myoblasts and augmented Myotube formation during differentiation, as assessed by increased Myotube length, aspect ratio, and the elevated mRNA expression of myogenic genes. Moreover, agrin treatment significantly increased acetylcholine receptor expression/clustering and Myotube formation and upregulated dystrophin expression in differentiated C2C12 Myotubes. Interestingly, the combination of topographical cues with agrin treatment further enhanced Myotube maturation and functionality as shown by improved contractility under electrical stimulation. Thus, combining topographical cues and agrin treatment improved functions of engineered muscle tissue, which has potential in biorobotics, drug screening, tissue engineering, and regenerative medicine.
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gelatin polyaniline composite nanofibers enhanced excitation contraction coupling system maturation in Myotubes
ACS Applied Materials & Interfaces, 2017Co-Authors: Serge Ostrovidov, Majid Ebrahimi, Hojae Bae, Hung K Nguyen, Sahar Salehi, Sang Bok Kim, Akichika Kumatani, Tomokazu Matsue, Xuetao Shi, Ken NakajimaAbstract:In this study, composite gelatin–polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced Myotube formation on composite gelatin–PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced Myotube maturation. Thus, in Myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation–contraction (E–C) coupling apparatus, calcium transients, and Myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation.
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Gelatin–Polyaniline Composite Nanofibers Enhanced Excitation–Contraction Coupling System Maturation in Myotubes
2017Co-Authors: Serge Ostrovidov, Majid Ebrahimi, Hojae Bae, Hung K Nguyen, Sahar Salehi, Sang Bok Kim, Akichika Kumatani, Tomokazu Matsue, Xuetao Shi, Ken NakajimaAbstract:In this study, composite gelatin–polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced Myotube formation on composite gelatin–PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced Myotube maturation. Thus, in Myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation–contraction (E–C) coupling apparatus, calcium transients, and Myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation
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Myotube formation on gelatin nanofibers multi walled carbon nanotubes hybrid scaffolds
Biomaterials, 2014Co-Authors: Serge Ostrovidov, Xiaobin Liang, Adnan Memic, Ken Nakajima, Faten Alhazmi, Mingwei Chen, Toshinori Fujie, Murugan Ramalingam, Ling Zhang, Ali KhademhosseiniAbstract:Engineering functional muscle tissue requires the formation of densely packed, aligned, and mature Myotubes. To enhance the formation of aligned Myotubes with improved contractibility, we fabricated aligned electrospun gelatin multi-walled carbon nanotubes (MWNTs) hybrid fibers that were used as scaffolds for the growth of myoblasts (C2C12). The MWNTs significantly enhanced Myotube formation by improving the mechanical properties of the resulting fibers and upregulated the activation of mechanotransduction related genes. In addition, the fibers enhanced the maturation of the Myotubes and the amplitude of the Myotube contractions under electrical stimulation (ES). Such hybrid material scaffolds may be useful to direct skeletal muscle cellular organization, improve cellular functionality and tissue formation.
Sahar Salehi - One of the best experts on this subject based on the ideXlab platform.
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Enhanced skeletal muscle formation on microfluidic spun gelatin methacryloyl (GelMA) fibres using surface patterning and agrin treatment.
Journal of tissue engineering and regenerative medicine, 2018Co-Authors: Majid Ebrahimi, Serge Ostrovidov, Hojae Bae, Sahar Salehi, Sang Bok Kim, Ali KhademhosseiniAbstract:Bioengineered functional muscle tissues are beneficial for regenerative medicine due to their treatment potential for various debilitating disorders, including myopathy and traumatic injuries. However, the contractile properties of engineered muscle constructs are lacking compared with their native counterparts. Here, we used microfluidic spinning to fabricate photocrosslinkable gelatin methacryloyl (GelMA) hydrogel fibres with well-defined surface morphologies for engineering muscle tissues. We examined whether the combination of topographical cues from surface micropatterning and biochemical stimulation with recombinant agrin can improve the generation of bioengineered muscle tissue. Topographical cues on micropatterned fibres promoted alignment of C2C12 myoblasts and augmented Myotube formation during differentiation, as assessed by increased Myotube length, aspect ratio, and the elevated mRNA expression of myogenic genes. Moreover, agrin treatment significantly increased acetylcholine receptor expression/clustering and Myotube formation and upregulated dystrophin expression in differentiated C2C12 Myotubes. Interestingly, the combination of topographical cues with agrin treatment further enhanced Myotube maturation and functionality as shown by improved contractility under electrical stimulation. Thus, combining topographical cues and agrin treatment improved functions of engineered muscle tissue, which has potential in biorobotics, drug screening, tissue engineering, and regenerative medicine.
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electrical stimulation of microengineered skeletal muscle tissue effect of stimulus parameters on Myotube contractility and maturation
Journal of Tissue Engineering and Regenerative Medicine, 2018Co-Authors: Ramin Banan Sadeghian, Majid Ebrahimi, Sahar SalehiAbstract:Skeletal muscle tissues engineered in vitro are aneural, are short in the number of fibres required to function properly and degenerate rapidly. Electrical stimulation has been widely used to compensate for such a lack of neural activity, yet the relationship between the stimulation parameters and the tissue response is subject to debate. Here we studied the effect of overnight electrical stimulation (training) on the contractility and maturity of aligned C2C12 Myotubes developed on micropatterned gelatin methacryloyl (GelMA) substrates. Bipolar rectangular pulse (BRP) trains with frequency, half-duration and applied pulse train amplitudes of f = 1 Hz, ton = 0.5 ms and Vapp = {3 V, 4 V, 4.5 V}, respectively, were applied for 12 h to the Myotubes formed on the microgrooved substrates. Aligned Myotubes were contracting throughout the training period for Vapp ≥ 4 V. Immediately after training, the samples were subjected to series of BRPs with 2 ≤ Vapp ≤ 5 V and 0.2 ≤ ton ≤ 0.9 ms, during which Myotube contraction dynamics were recorded. Analysis of post-training contraction revealed that only the Myotubes trained at Vapp = 4 V displayed consistent and repeatable contraction profiles, showing the dynamics of Myotube contractility as a function of triggering pulse voltage and current amplitudes, duration and imposed electrical energy. In addition, Myotubes trained at Vapp = 4 V displayed amplified expression levels of genes pertinent to sarcomere development correlated with Myotube maturation. Our findings are imperative for a better understanding of the influence of electrical pulses on the maturation of microengineered Myotubes.
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gelatin polyaniline composite nanofibers enhanced excitation contraction coupling system maturation in Myotubes
ACS Applied Materials & Interfaces, 2017Co-Authors: Serge Ostrovidov, Majid Ebrahimi, Hojae Bae, Hung K Nguyen, Sahar Salehi, Sang Bok Kim, Akichika Kumatani, Tomokazu Matsue, Xuetao Shi, Ken NakajimaAbstract:In this study, composite gelatin–polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced Myotube formation on composite gelatin–PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced Myotube maturation. Thus, in Myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation–contraction (E–C) coupling apparatus, calcium transients, and Myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation.
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Gelatin–Polyaniline Composite Nanofibers Enhanced Excitation–Contraction Coupling System Maturation in Myotubes
2017Co-Authors: Serge Ostrovidov, Majid Ebrahimi, Hojae Bae, Hung K Nguyen, Sahar Salehi, Sang Bok Kim, Akichika Kumatani, Tomokazu Matsue, Xuetao Shi, Ken NakajimaAbstract:In this study, composite gelatin–polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced Myotube formation on composite gelatin–PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced Myotube maturation. Thus, in Myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation–contraction (E–C) coupling apparatus, calcium transients, and Myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation
Ken Nakajima - One of the best experts on this subject based on the ideXlab platform.
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gelatin polyaniline composite nanofibers enhanced excitation contraction coupling system maturation in Myotubes
ACS Applied Materials & Interfaces, 2017Co-Authors: Serge Ostrovidov, Majid Ebrahimi, Hojae Bae, Hung K Nguyen, Sahar Salehi, Sang Bok Kim, Akichika Kumatani, Tomokazu Matsue, Xuetao Shi, Ken NakajimaAbstract:In this study, composite gelatin–polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced Myotube formation on composite gelatin–PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced Myotube maturation. Thus, in Myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation–contraction (E–C) coupling apparatus, calcium transients, and Myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation.
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Gelatin–Polyaniline Composite Nanofibers Enhanced Excitation–Contraction Coupling System Maturation in Myotubes
2017Co-Authors: Serge Ostrovidov, Majid Ebrahimi, Hojae Bae, Hung K Nguyen, Sahar Salehi, Sang Bok Kim, Akichika Kumatani, Tomokazu Matsue, Xuetao Shi, Ken NakajimaAbstract:In this study, composite gelatin–polyaniline (PANI) nanofibers doped with camphorsulfonic acid (CSA) were fabricated by electrospinning and used as substrates to culture C2C12 myoblast cells. We observed enhanced Myotube formation on composite gelatin–PANI nanofibers compared to gelatin nanofibers, concomitantly with enhanced Myotube maturation. Thus, in Myotubes, intracellular organization, colocalization of the dihydropyridine receptor (DHPR) and ryanodine receptor (RyR), expression of genes correlated to the excitation–contraction (E–C) coupling apparatus, calcium transients, and Myotube contractibility were increased. Such composite material scaffolds combining topographical and electrically conductive cues may be useful to direct skeletal muscle cell organization and to improve cellular maturation, functionality, and tissue formation
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Myotube formation on gelatin nanofibers multi walled carbon nanotubes hybrid scaffolds
Biomaterials, 2014Co-Authors: Serge Ostrovidov, Xiaobin Liang, Adnan Memic, Ken Nakajima, Faten Alhazmi, Mingwei Chen, Toshinori Fujie, Murugan Ramalingam, Ling Zhang, Ali KhademhosseiniAbstract:Engineering functional muscle tissue requires the formation of densely packed, aligned, and mature Myotubes. To enhance the formation of aligned Myotubes with improved contractibility, we fabricated aligned electrospun gelatin multi-walled carbon nanotubes (MWNTs) hybrid fibers that were used as scaffolds for the growth of myoblasts (C2C12). The MWNTs significantly enhanced Myotube formation by improving the mechanical properties of the resulting fibers and upregulated the activation of mechanotransduction related genes. In addition, the fibers enhanced the maturation of the Myotubes and the amplitude of the Myotube contractions under electrical stimulation (ES). Such hybrid material scaffolds may be useful to direct skeletal muscle cellular organization, improve cellular functionality and tissue formation.
