Effects Of Mechanical Stretch On The Alignment And Differentiation Of The Multilayer Myotubes Cultured In Vitro

Skeletal muscle is a kind of musclefiberbundle constituted by parallel myotubes. Myotubes’synergy completes the systolic function; Various reasons cause the defection and the loss of function, such as congenital defect, injury, tumor, primary myopathy and metabolic diseases. Traditional treatment method is mainly about transplantation of myocutaneous flap. But the limitation of available muscle sources, the damage of donators, and immunological rejection inhibit the application of myocutaneous flap. In recent years, the researchers try to use myoblast transplantation to promote muscle fiber regeneration and treat genetic myopathy. But there are some problems such as uneven distribution of myoblasts in the body, low survival rate and immune rejection. The development of the skeletal muscle tissue engineering bring the repairation of the structure and function hope. The culture of parallel myotubes in vitro and the maintenance of muscle activity are the key point of the muscle tissue regeneration. In recent years skeletal muscle tissue engineering focuses on how to optimize the myoblasts culture conditions in vitro to get more functional parallel myotubes. The researchers made progress on the study of three-dimensional scaffolds, directional magnetic and electrical and mechanical stretch stimulate. Our organs are in complex growth environment. The mechanical stretch greatly influence the form of structure and function. Persistent passive tension in human body promote skeletal muscle, make skeletal muscle thicker and longer. Physical labor and exercise can also increase the amount and diameter of muscle. In physiological condition, mechanical stretch is an important stimulation, maintaining cell survival and growth. It affects the growth of cells behavior, adjusts the cell metabolism and gene expression process. It plays an important role in cell proliferation and differentiation. Research shows that the mechanical stretch loading into muscle cells have bidirectional effect. The suitable tensile can promote cell proliferation, while excessive stretch makes cells apoptosis. How does the signal of mechanical stimulation affect the growth and development? Which way does the mechanical stimulation change the cells growth condition? The above two questions have become a research focus in the mechanics in recent years. It has been confirmed that, the periodic mechanical loading for regeneration muscle tissue, simulating the growth process of skeletal muscle tissue in the body, can significantly affect the activity survival cycle and systolic function of regeneration muscle tissue.In vitro culture conditions, obtaining parallel myotubes is the key of building muscle tissue model to success. The parallel arrangement characteristic of mature muscle tissue, is the morphological basis of contraction function. Sylgard184belongs to silicone rubber elastic material. after the mixed two-component curing, it becomes flexible and smooth surface. And it’s convenient of the mechanical stretch for parallel myotubes which growth on the elastic matrix materials. On the early stage of the study, we made preliminary exploration in the construct of three-dimensional regenerative skeletal muscle tissue in vitro. We used Sylgard184as a cell matrix to culture with C2C12cells. And finally we got the mature parallel myotubes. Based on the early study, we treated the myoblasts with various mechanical stretch applied with cells tensile instrument, and then compare the difference of themorphological characteristics and myotubes’phenotypic differentiation which are in different mechanical loading conditions, select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro. In addition, we use homemade cells instrument and Flexcell tensile mechanical loading system stretch the myotubes which are cultured in three-dimensional and plane condition respectively. Comparing the two methods, we found that three-dimensional culture condition and mechanical stretch are very important for mature three-dimensional regenerative skeletal muscle tissue in vitro.Three parts of our research.Part one:Construction and detection of the alignment and differentiation of the multilayer myotubes cultured in vitroObjective:To cultivate C2C12cells on the modified mold-grooves of Sylgard184and induce C2C12cells differentiation to highly organized3-dimensional muscle tissue,then detect the growth and functional properties of differentiation myotube. Methods:Mix bicomponents Sylgard184by the ratio of10:1uniformly and poured the mixture into6-well plates. After curing by standing at room temperature(The width of each mold groove is0.8mm, depth is0.15mm, length can adjust by requiring), carve grooves at the central basal Sylgard184and indentate in the groove at the bottom of the mold. After washing grooves with PBS fluid, pave matrigel matrix and collagen (mixing ratio1:6) mixture evenly at the bottom of grooves and add0.3ml mixture into each groove, making sure that the cell matrix material of coverage is uniform. Finally, place them in biological safety cabinet and under UV disinfection. Inoculate C2C12cell suspension whose density is1.0～2.0×105/ml, remove the entire medium when there are80%cells proliferations, then add2%horse serum containing DMEM/F12and differentiation cultivate7d, the inverted microscope to observe X-ray film morphology and continue to divide after the10d, the construction of skeletal muscle tissue was detected by immunofluorescence assay muscle-specific protein expression. RT-PCR was used to detect Myogenin, Desmin, MyHC mRNAs expression, while the use of electron microscopy to watch myotubes’morphologies and inter-connections.Results:C2C12cells were differentiation cultivated in the mold-grooves of Sylgard184for7days, polarity-differentiation and myotube fusion were observed under the inverted microscope. After10days, it showed that myotubes stand closely and connect with each other deteced by the scanning electron microscopy.The thickness of the membrane-like structure with the character of three-dimensional was0.15mm. Detect Myogenin. Desmin, MyHC mRNAs of positive expression in constructed skeletal muscle tissue by RT-PCR, and there is differentiation-specific gene protein Desmin, MyHC myoblast-specific protein expression by immunofluorescence assay and DAPI nuclear staining.Results show that desmin are skeletal muscle cytoskeleton protein and intermediate filament-specific protein, which mainly distributed in the cytoplasm of skeletal muscle cells, and are also one of the earliest indication of skeletal muscle. It expresses in the differentiated myotube in our results, which indicated that the constructed three-dimensional differentiated muscle tissue polarity analogues have the same physiological characteristics with mature skeletal muscles in vivo.Conclusions:The modified mold-grooves of Sylgard184is able to guide the differentiation of C2C12cells and promotes them to form parallel multinucleated myotubes. Myotubes could overlap with each other and form three-dimensional skeletal muscle tissue. Part two:Effects of mechanical stretch with variant frequencies on the alignment and differentiation of the multilayer myotubes cultured in vitro Objective:To explore the effects of mechanical stretch with variant frequencies on the alignment and differentiation of the multilayer myotubes cultured in vitro, to select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro.Methods:C2C12myoblasts cultured in the groove casts of Sylgard184were induced into the multilayer myotubes cultured in vitro. Meanwhile we treated the myoblasts with various mechanical stretch applied with cells tensile instrument. The myotubes morphology were observed by inverted microscope. And the expressions of mRNA for MyoD, Myogenin, Desmin and MyHC was detected by RT-PCR and qPCR, respectively.Results:The mechanical stretch could promote the aligned fusion and increase the number of myotubes. Indeed the multilayer myotubes aligned more closely in the condition of mechanical stretch with the amplitude of10%and the frequency of0.25Hz for7days. What’s more, the expressions of mRNA for Myogenin, Desmin and MyHC could be increased by mechanical stretch. But in the group with the frequency of1.00Hz or stretch for10days mechanical stretch accelerated multilayer myotubes’ death and exfoliation and also decreased the expression of mRNA for MyoD, Myogenin, Desmin and MyHC.Conclusions:Low frequency and timely periodic mechanical stretch is beneficial to the differentiation of the multilayer myotubes cultured in the groove casts of Sylgard184, but as the stretch time goes on the aging of myotubes will be accelerated.Part three:Comparative study of effects of mechanical stretch on the alignment and differentiation of myotubes cultured in three-dimensional and plane scaffoldsObjective:To compare the differences of effects of mechanical stretch on the alignment and differentiation of myotubes cultured between in three-dimensional and in plane scaffolds. To explore the importance of polar growth for the Construction of organized3-dimensional muscle tissueMethods:C2C12myoblasts cultured in the BioFlex plates were induced into the multilayer myotubes cultured in vitro, called group A. The myoblasts were treated with mechanical stretch applied by Flex cell instrument, called group B. Meanwhile C2C12myoblasts cultured in the groove casts of Sylgard184were induced into the multilayer myotubes, called group C, which were treated with the same mechanical stretch applied by Cells tensile instrument, called group D. The myotubes’ morphology of all the groups were observed by inverted microscope. And the expressions of mRNA for Myogenin, Desmin and MyHC was detected by RT-PCR and qPCR, respectively. Immunofluorescence detected the protein expression of MyHC.Results:Mechanical stretch could promote the aligned fusion of myotubes. Compared with that of plane culture, the three-dimensional culture could promote the aligned fusion and increase the number of myotubes, no matter with mechanical stretch or not. The greatest mRNA expression of Myogenin, Desmin. MyHC were found in group D, and the lowest expression in group B.Conclusions:Compared with that of plane culture, the three-dimensional culture could promote the alignment and differentiation of myotubes, low frequency and timely periodic mechanical stretch could enhance the promotion. Although mechanical stretch can promote the alignment of myotubes, it also inhibits the differentiation of myotubes cultured in the BioFlex plates.