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1.
J Biosci Bioeng ; 132(4): 417-422, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34348874

RESUMO

Electric pulse-stimulated C2C12 myotubes are gaining interest in the field of muscle physiology and biotechnology because electric pulse stimulation (EPS) enhances sarcomere structure development and active tension generation capability. Recently, we found that termination of EPS results in the rapid loss of active tension generation accompanied by disassembly of the sarcomere structure, which may represent an in vitro muscle atrophy model. To elucidate the molecular mechanism underlying this rapid loss of active tension generation and sarcomere structure disassembly after termination of EPS, we performed transcriptomic analysis using microarray. After termination of EPS, 74 genes were upregulated and 120 genes were downregulated after 30 min; however, atrophy-related genes were not found among these genes. To further assess the effect of EPS on gene expression, we re-applied EPS after its termination for 8 h and searched for genes whose expression was reversed. Four genes were upregulated by termination of EPS and downregulated by the re-application of EPS, whereas two genes were downregulated by termination of EPS and upregulated by the re-application of EPS. Although none of these genes were atrophy- or hypertrophy-related, the results presented in this study will contribute to the understanding of gene expression changes that mediate rapid loss of active tension generation and sarcomere structure disassembly following termination of EPS in C2C12 myotubes.

2.
Nat Commun ; 12(1): 5059, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429413

RESUMO

With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats.


Assuntos
Bioimpressão/métodos , Géis , Carne , Tendões , Animais , Bovinos , Técnicas de Cultura de Células , Colágeno , Células Endoteliais , Músculos/citologia , Músculos/fisiologia , Impressão Tridimensional , Células-Tronco , Tendões/citologia , Engenharia Tecidual
3.
J Biosci Bioeng ; 131(5): 572-578, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33422389

RESUMO

We have studied the effects of hydrogen peroxide (H2O2) on the differentiation and maintenance of C2C12 myoblasts. The effects of H2O2 were evaluated by cell viability, total protein concentration, the relative amount of muscle-related proteins, sarcomere structure, and active tension generation. Oxidative stress is one of the major causes of myopathy after exercise and thus establishing the method to evaluate the effects on muscle function is essential. The primary function of striated muscle is to generate force, thus, the measurement of active tension is important in assessing the effect of chemicals on muscle. Among the indices we tested, the sarcomere structure was the most sensitive to the H2O2 exposure while the cell viability was less sensitive. The effects of H2O2 on active tension correlated with a decrease in the amount of muscle proteins. In this study, our results showed that the effect of chemicals on muscle should be measured in multiple ways, including active tension generation, for a better understanding of its physiological impact.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Músculo Esquelético/citologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos
4.
Appl Environ Microbiol ; 86(14)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414798

RESUMO

Many phenylalanine- and tyrosine-producing strains have used plasmid-based overexpression of pathway genes. The resulting strains achieved high titers and yields of phenylalanine and tyrosine. Chromosomally engineered, plasmid-free producers have shown lower titers and yields than plasmid-based strains, but the former are advantageous in terms of cultivation cost and public health/environmental risk. Therefore, we engineered here the Escherichia coli chromosome to create superior phenylalanine- and tyrosine-overproducing strains that did not depend on plasmid-based expression. Integration into the E. coli chromosome of two central metabolic pathway genes (ppsA and tktA) and eight shikimate pathway genes (aroA, aroB, aroC, aroD, aroE, aroGfbr , aroL, and pheAfbr ), controlled by the T7lac promoter, resulted in excellent titers and yields of phenylalanine; the superscript "fbr" indicates that the enzyme encoded by the gene was feedback resistant. The generated strain could be changed to be a superior tyrosine-producing strain by replacing pheAfbr with tyrAfbr A rational approach revealed that integration of seven genes (ppsA, tktA, aroA, aroB, aroC, aroGfbr , and pheAfbr ) was necessary as the minimum gene set for high-yield phenylalanine production in E. coli MG1655 (tyrR, adhE, ldhA, pykF, pflDC, and ascF deletant). The phenylalanine- and tyrosine-producing strains were further applied to generate phenyllactic acid-, 4-hydroxyphenyllactic acid-, tyramine-, and tyrosol-producing strains; yield of these aromatic compounds increased proportionally to the increase in phenylalanine and tyrosine yields.IMPORTANCE Plasmid-free strains for aromatic compound production are desired in the aspect of industrial application. However, the yields of phenylalanine and tyrosine have been considerably lower in plasmid-free strains than in plasmid-based strains. The significance of this research is that we succeeded in generating superior plasmid-free phenylalanine- and tyrosine-producing strains by engineering the E. coli chromosome, which was comparable to that in plasmid-based strains. The generated strains have a potential to generate superior strains for the production of aromatic compounds. Actually, we demonstrated that four kinds of aromatic compounds could be produced from glucose with high yields (e.g., 0.28 g tyrosol/g glucose).


Assuntos
Bactérias/metabolismo , Cromossomos Bacterianos/genética , Engenharia Genética , Fenilalanina/metabolismo , Tirosina/metabolismo , Escherichia coli/genética , Plasmídeos/genética
5.
J Biosci Bioeng ; 130(1): 98-105, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32278672

RESUMO

Although various types of artificial skeletal muscle tissue have been reported, the contractile forces generated by tissue-engineered artificial skeletal muscles remain to be improved for biological model and clinical applications. In this study, we investigated the effects of extracellular matrix (ECM) and supplementation of a small molecule, which has been reported to enhance α7ß1 integrin expression (SU9516), on cell migration speed, cell fusion rate, myoblast (mouse C2C12 cells) differentiation and contractile force generation of tissue-engineered artificial skeletal muscles. When cells were cultured on varying ECM coated-surfaces, we observed significant enhancement in the migration speed, while the myotube formation (differentiation ratio) decreased in all except for cells cultured on Matrigel coated-surfaces. In contrast, SU9516 supplementation resulted in an increase in both the myotube width and differentiation ratio. Following combined culture with a Matrigel-coated surface and SU9516 supplementation, myotube width was further increased. Additionally, contractile forces produced by the tissue-engineered artificial skeletal muscles was augmented following combined culture. These findings indicate that regulation of the cell-ECM interaction is a promising approach to improve the function of tissue-engineered artificial skeletal muscles.


Assuntos
Matriz Extracelular/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Colágeno/metabolismo , Combinação de Medicamentos , Integrinas/genética , Integrinas/metabolismo , Laminina/metabolismo , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteoglicanas/metabolismo
6.
J Biosci Bioeng ; 126(5): 586-595, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29958770

RESUMO

Many metabolic engineering approaches have been attempted to generate strains capable of producing valuable compounds. One of main goals is industrial application of these strains. Integration of synthetic pathway genes into the Escherichia coli chromosome enables generation of a plasmid-free strain that is stable and useful for industrial applications. Strains that do not require induction are advantageous in terms of cost. In the present study, we constructed a constitutive overexpression system in E. coli to generate plasmid-free and inducer-free strains. The T7 RNA polymerase/T7 promoter overexpression system, which is an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible gene overexpression system (T7-dependent inducible overexpression system), was modified to be a constitutive overexpression system. The constructed overexpression system, a "chromosome-based T7-dependent constitutive overexpression system", was applied in a metabolic engineering study to generate a plasmid-free and inducer-free phenylalanine producing strain of E. coli.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Fenilalanina/metabolismo , Cromossomos Bacterianos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas/genética , Organismos Geneticamente Modificados , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
J Biosci Bioeng ; 124(4): 469-475, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28601606

RESUMO

Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 ± 0.4) × 101-(2.8 ± 0.5) × 101 cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 ± 0.8) × 101-(6.8 ± 1.1) × 102 cells/aggregate) during early-stage of culture (24-72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 ± 0.2) × 103-(3.5 ± 1.1) × 103 cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72-120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate.


Assuntos
Agregação Celular , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Agregação Celular/efeitos dos fármacos , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Tamanho Celular , Terapia Baseada em Transplante de Células e Tecidos , Colágeno Tipo I/metabolismo , Meios de Cultura/farmacologia , Humanos , Fatores de Tempo
8.
J Biosci Bioeng ; 123(2): 259-264, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27590346

RESUMO

Rhabdomyosarcoma (RMS) is a highly malignant tumor type of skeletal muscle origin, hallmarked by local invasion. Interaction between invasive tumor cells and normal cells plays a major role in tumor invasion and metastasis. Culturing tumor cells in a three-dimensional (3D) model can translate tumor malignancy relevant cell-cell interaction. To mimic tumor heterogeneity in vitro, a co-culture system consisting of a malignant embryonal rhabdomyosarcoma (ERMS) cell line RD and a normal human skeletal muscle myoblast (HSMM) cell line was established by cell sheet technology. Various ratios of RDs to HSMMs were employed to understand the quantitative effect on intercellular interactions. Disruption of sheet structure was observed in heterogeneous cell sheets having a low ratio of RDs to HSMMs, whereas homogeneous HSMM or RD sheets maintained intact structure. Deeper exploration of dynamic tumor cell behavior inside HSMM sheets revealed that HSMM cell alignment was disrupted by highly motile RDs. This study demonstrated that RMS cells are capable of compromising their surrounding environment through induced decay of HSMMs alignment in a cell-based 3D system. This suggests that muscle disruption might be a major consequence of RMS cell invasion into muscles, which could be a promising target to preventing tumor invasion.


Assuntos
Comunicação Celular , Movimento Celular , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/fisiologia , Rabdomiossarcoma/patologia , Adesão Celular , Técnicas de Cultura de Células/métodos , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura/métodos , Humanos , Junções Intercelulares/patologia , Junções Intercelulares/fisiologia , Invasividade Neoplásica , Migração Transendotelial e Transepitelial
9.
Bioprocess Biosyst Eng ; 40(1): 123-131, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27638317

RESUMO

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 106 cells mL-1 was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.


Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Diálise , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
10.
Sci Rep ; 4: 4781, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24759171

RESUMO

Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.


Assuntos
Estimulação Elétrica , Músculo Esquelético/fisiologia , Engenharia Tecidual , Animais , Células Cultivadas , Camundongos , Contração Muscular/fisiologia , Mioblastos/fisiologia , Fatores de Tempo , Técnicas de Cultura de Tecidos , Engenharia Tecidual/métodos
11.
Biotechnol Lett ; 35(7): 1001-8, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23515892

RESUMO

Using a cell sheet stacking method, we developed an in vitro culture system in which green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVECs) were cultured under human skeletal muscle myoblast (HSMM) sheets with different layer numbers. Our aim in developing this system was to examine the different endothelial behaviors in the cell sheet. During 96 h of incubation, in monolayer HSMM sheet, HUVECs quickly reached the top of the cell sheet and detached. In three-layered HSMM sheet, HUVECs also migrated to the top layer and formed island-shaped aggregates. In five-layered HSMM sheet, HUVECs migrated into the middle of the cell sheet and formed net-shaped aggregates. In seven-layered HSMM sheet, HUVECs migrated in the basal of the cell sheet and formed sparse net-shaped aggregates. The thickness of the HSMM sheet, which can be controlled by the layer number of the cell sheet, is therefore an important parameter that affects the migration time, encounters, localization, and morphology of HUVECs inside the HSMM sheet.


Assuntos
Células Endoteliais/fisiologia , Mioblastos/fisiologia , Técnicas de Cultura de Células , Movimento Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas de Cultura de Órgãos
12.
Bioprocess Biosyst Eng ; 36(9): 1261-5, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23223911

RESUMO

In the present study, to elucidate mechanisms of growth suppression in YIBO-pdc1/5Δ, we performed carbon metabolic flux analysis under micro-aerobic conditions. Our results indicate that growth suppression of YIBO-pdc1/5Δ is caused by decreased flux to the pentose phosphate pathway, which supplies ribose-5-phosphate, a precursor for histidine synthesis in Sacchar omyces cerevisiae. In addition, significant accumulation of pyruvate was observed in the continuous culture.


Assuntos
Ácido Láctico/biossíntese , Via de Pentose Fosfato , Saccharomyces cerevisiae/metabolismo , Aerobiose/genética , Engenharia Genética , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/genética
13.
J Biosci Bioeng ; 115(2): 193-5, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23021912

RESUMO

A simple fed-batch system for cultivating genetically engineered yeast generating lactate under the regulation of the PDC1 promoter was established. Traditional strategies that avoid occurrence of Crabtree effect, such as respiratory quotient (RQ) control or ethanol control, are not applicable to the strain because of reduced generation of ethanol and CO(2) by-products. In this system, the feed rate increased when the pH was >5.0, and decreased when the pH was <5.0. Using this system, cell yields on sucrose increased by approximately 30% compared to that with the conventional RQ control method, due to the early detection of occurrence of Crabtree effect by pH decrease.


Assuntos
Reatores Biológicos , Fermentação , Engenharia Genética , Ácido Láctico/metabolismo , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Dióxido de Carbono/metabolismo , Etanol/metabolismo , Concentração de Íons de Hidrogênio , Engenharia Metabólica , Piruvato Descarboxilase/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Sacarose/metabolismo , Fatores de Tempo
14.
J Biosci Bioeng ; 115(2): 115-21, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23026451

RESUMO

Skeletal muscle is the most abundant tissue in the body, and its capability of generating an active force is one of the most significant features. In order to study the physiology and disorders related to the skeletal muscle using cells in vitro, the active force should be evaluated, in addition to molecular and cell biological experiments performed. This article reviews an evaluation system for the active tension generated by cultured skeletal muscle cells or tissue-engineered skeletal muscles. First, we focused on experimental models involving a single or several myotubes. Then, we focused on the systems for tissue-engineered skeletal muscles consisting of much larger numbers of myotubes. Such systems can be used to study the physiology of the skeletal muscle, screen drug candidates for skeletal muscle-related disorders, and improve the function of tissue-engineered skeletal muscle.


Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/patologia , Engenharia Tecidual
15.
Biomaterials ; 34(3): 662-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23117213

RESUMO

Autologous transplantation of myoblast sheet has attracted attention as a new technique for curing myocardial infarction. Myoblast sheet has the ability to secret cytokines that improve heart function via the facilitation of angiogenesis on affected part. To mimic the in vivo angiogenesis in the myoblast sheet after transplantation, a five-layered cell sheet of human skeletal muscle myoblasts (HSMMs) was overlaid on human umbilical vein endothelial cells (HUVECs) which enables evaluation of dynamic HUVEC behavior. HUVECs existing initially at the bottom of the sheet changed to be a stretched shape and migrated upward compared with the surrounding HSMMs in the sheet. Prolonged incubation resulted in network formation of HUVECs in the middle of the sheet, although non-networked HUVECs continued to migrate to the top of the sheet, which meant the spatial habitation of HUVECs in the cell sheet. Image processing was performed to determine the variation in the extent of network formation at different HUVEC densities. It was found that the extent of formed network depended on the frequency of encounters among HUVECs in the middle of the sheet. The present system, which can evaluate network formation, is considered to be a promising in vitro angiogenesis model.


Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Mioblastos Esqueléticos/citologia , Neovascularização Fisiológica , Engenharia Tecidual/métodos , Movimento Celular , Humanos
16.
J Biosci Bioeng ; 113(1): 128-31, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22018737

RESUMO

The procedure for fabricating a multilayered cell sheet has been developed by combining multiple sheets using a thermo-responsive surface and stamp system. Confocal laser scanning microscopy revealed that the fluidity of a multilayered sheet of skeletal myoblasts could be estimated as vertical diffusivity and changed upon addition of dermal fibroblasts.


Assuntos
Fibroblastos/citologia , Mioblastos Esqueléticos/citologia , Engenharia Tecidual/métodos , Movimento Celular , Células Cultivadas , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Temperatura
17.
J Biosci Bioeng ; 112(3): 273-8, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21646045

RESUMO

The aim of this study was to investigate whether insulin-like growth factor (IGF)-I gene delivery to myoblast cells promotes the contractile force generated by hydrogel-based tissue-engineered skeletal muscles in vitro. Two retroviral vectors allowing doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into mouse myoblast C2C12 cells to evaluate the effects of IGF-I gene expression on these cells. IGF-I gene expression stimulated the proliferation of C2C12 cells, and a significant increase in the growth rate was observed for IGF-I-transduced C2C12 cells with Dox addition, designated C2C12/IGF (Dox+) cells. Quantitative morphometric analyses showed that the myotubes induced from C2C12/IGF (Dox+) cells had a larger area and a greater width than control myotubes induced from normal C2C12 cells. Artificial skeletal muscle tissues were prepared from the respective cells using hydrogels composed of type I collagen and Matrigel. Western blot analyses revealed that the C2C12/IGF (Dox+) tissue constructs showed activation of a skeletal muscle hypertrophy marker (Akt) and enhanced expression of muscle-specific markers (myogenin, myosin heavy chain and tropomyosin). Moreover, the creatine kinase activity was increased in the C2C12/IGF (Dox+) tissue constructs. The C2C12/IGF (Dox+) tissue constructs contracted in response to electrical pulses, and generated a significantly higher physical force than the control C2C12 tissue constructs. These findings indicate that IGF-I gene transfer has the potential to yield functional skeletal muscle substitutes that are capable of in vivo restoration of the load-bearing function of injured muscle or acting as in vitro electrically-controlled bio-actuators.


Assuntos
Fator de Crescimento Insulin-Like I/genética , Músculo Esquelético/citologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Colágeno , Combinação de Medicamentos , Vetores Genéticos , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Laminina , Camundongos , Contração Muscular , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Fosforilação , Proteoglicanas , Somatomedinas/metabolismo , Suporte de Carga
18.
Biomed Microdevices ; 13(1): 123-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20957437

RESUMO

With the aim of designing a mechanical drug delivery system involving a bio-actuator, we fabricated a Micro Electro Mechanical Systems (MEMS) device that can be driven through contraction of skeletal muscle cells. The device is composed of a Si-MEMS with springs and ratchets, UV-crosslinked collagen film for cell attachment, and C2C12 muscle cells. The Si-MEMS device is 600 µm x 1000 µm in size and the width of the collagen film is 250 ~ 350 µm, which may allow the device to go through small blood vessels. To position the collagen film on the MEMS device, a thermo-sensitive polymer was used as the sacrifice-layer which was selectively removed with O2 plasma at the positions where the collagen film was glued. The C2C12 myoblasts were seeded on the collagen film, where they proliferated and formed myotubes after induction of differentiation. When C2C12 myotubes were stimulated with electric pulses, contraction of the collagen film-C2C12 myotube complex was observed. When the edge of the Si-MEMS device was observed, displacement of ~8 µm was observed, demonstrating the possibility of locomotive movement when the device is placed on a track of adequate width. Here, we propose that the C2C12-collagen film complex is a new generation actuator for MEMS devices that utilize glucose as fuel, which will be useful in environments in which glucose is abundant such as inside a blood vessel.


Assuntos
Fenômenos Mecânicos , Microtecnologia/instrumentação , Fibras Musculares Esqueléticas/citologia , Integração de Sistemas , Animais , Linhagem Celular , Colágeno/metabolismo , Estimulação Elétrica , Metabolismo Energético , Glucose/metabolismo , Camundongos , Fibras Musculares Esqueléticas/metabolismo
19.
Tissue Eng Part A ; 17(1-2): 107-14, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20672996

RESUMO

Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2 µN [1.06 mN/mm2]). Rheobase and chronaxie of the tissue were determined as 4.45 V and 0.72 ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies.


Assuntos
Magnetismo , Músculo Esquelético , Engenharia Tecidual/métodos , Animais , Western Blotting , Linhagem Celular , Camundongos , Miogenina/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Tropomiosina/metabolismo
20.
J Muscle Res Cell Motil ; 31(4): 279-88, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21120590

RESUMO

We found that the active tension of C2C12 myotubes that had been subjected to artificial exercise for ~10 days decreased rapidly after termination of the artificial exercise. When differentiated C2C12 myotubes were subjected to continuous 1 Hz artificial exercise for ~10 days, the active tension increased to ~4× compared to that before application of the artificial exercise, as reported previously. On termination of artificial exercise, the active tension decreased rapidly, the level reaching that before application of the artificial exercise within 8 h. Concomitant with the decrease in the active tension, an increase in the amount of ubiquitinated proteins was observed. Real time RT-PCR revealed that the expression of several genes associated with atrophy, namely Smc6, Vegfa, Jarid2, Kitl, Cds2, Inmt, Fasn, Neurl, Topors, and Cul2, were also changed after termination of artificial exercise. These results indicate that termination of artificial exercise induced atrophy-like responses of C2C12 myotubes. Here we found that during the decrease in active tension, the sarcomere structure, especially the thin filament structure, decayed rapidly after termination of artificial exercise. On reapplication of the artificial exercise, the active tension was restored rapidly, within 8 h, concomitant with reformation of the sarcomere structure. These results indicate that disassembly of the sarcomere structure may be one of the reasons for the active tension decrease during disuse muscle atrophy.


Assuntos
Fibras Musculares Esqueléticas/fisiologia , Tono Muscular , Condicionamento Físico Animal/métodos , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcômeros/fisiologia , Sarcômeros/ultraestrutura , Fatores de Tempo
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