Prelimilary Experimental Study Of Three-demensional Engineered Skeletal Muscle Tissue In Vitro

The development of skeletal muscle tissue engineering will provide some treating advice for many skeletal muscle diseases such as muscular dystrophy,spinal muscular atrophy,etc.Besides,plastic surgery for the treatment of trauma,tumor resection and denervation-induced loss of muscle tissue will also benefit from skeletal muscle tissue engineering.Even though tissue engineering research has made significant progress in many artificial tissues such as skin,bone and cartilage have been used in clinical treatements,but skeletal muscle tissue engineering still faces immense challenges and it has difficulties in the high density at the cell proliferation and cell differentiation,three-dimensional polar culture,construction of the larger the size of the muscle tissue,etc.However,many scholars are still committed to skeletal muscle in vitro and alternative research,attempt to create a function of the muscle tissue. After many years of exploration and efforts,a number of authoritative research team made considerable achievements,and constantly push forward the study of skeletal muscle tissue engineering progress:Powell^[1,2] used collagen and matrix to build a successful three-dimensional skeletal muscle and added mechanical stimulation to tissue,making sure the formation of muscle tissue,differentiation and the formation of contractile function in vitro;Dennis and Kosnik^[3,4] explored three-dimensional skeletal self organized model and added electric stimulation to the muscle tissue Myooids in the horizonl it would produce a sustained contraction of the excitatory and function.In Germany Bach's team^[5] had co-cultured myoblasts fibrin and scaffold in three-dimension and obtained myotubes that had the multi-polar differentiated functional.The key technologies of skeletal muscle construction in vitro include implanting largly amplified in vitro target cells such as adult skeletal muscle satellite cells or myoblast cell line into the three-dimensional matrix of different materials and making them grow and differentiate into skeletal muscle in vitro analogues that have shapes and contraction ability and could survive in a long-term.To realize the true meaning of skeletal muscle in vitro construction,foreign scholars have improved skeletal muscle tissue construction technology,at the same time,detect expression of key regulatory molecules(such as the myogenic transcription factors,contractile proteins, muscle creatine kinase,acetylcholine receptor,etc.) systematically in muscle tissues that differentiate in vitro and composite with different matrix.They have tried to explore whether the differentiation,the corresponding expression of specific transcription factors and control of the signal system in muscle tissues in vitro are coincided with growth and differentiation of the body's skeletal myoblast.The results of above skeletal muscle construction study provide clues and ideas for future skeletal muscle tissue engineering researchers.Through analyzing home and abroad skeletal muscle research data we found that though it is still difficult to construct in vitro large skeletal muscles that can mimic the features and function of structure of muscle in vivo,the existing and exploring in vitro construction muscle tissues are able to mimic the differentiation and development of in vivo tissues, express contractile proteins,produce contraction effect and avoid the interference from other cells or tissues,making them become ideal in vitro model of studying the development of skeletal muscle and skeletal muscle myopathy.However,there are no coverages of success three-dimensional construction of muscle tissue in vitro at home. Sylgard 184 belongs to silicone rubber elastic material and has two-component mixed-curing,solidification at room temperature,the surface of it is smooth and has a certain degree of flexibility.As a basal layer,benift to detect the cell'movement and traction force generated by load^[6].Therefore,Sylgard 184 has been widely used in skeletal muscle tissue engerring,and more scholars pay attention to it^[7,8,9].In view of the above research background,this study selected Sylgard 184 (silicone elastic material) as the growth of stents that co-cultured with myoblasts in vitro and explored the practicability of constructing in vitro three-dimensional model of skeletal muscle.As the effective contact of scaffold material and seed cells decides cell proliferation,differentiation and the function expression we first coate Sylgard184 surface with different matrix components(collagen,laminin,etc.),and then analyze growth and differentiation of myoblasts at different matrix materials Sylgard184 surface-modified,finally,select the best surface-modified ingredients;As the keys to construction of three-dimensional muscle tissue in vitro,are emergence and maintenance of polar parallel differentiated myotube,this study further modified to optimize the Sylgard184 indentation to the mold through the guide groove cast,as well as promote growth and scalability of the Matrigel matrix,obtain the polar parallel differentiated muscle tissue,and analyze and detect muscle characteristics and systolic function of polarity of muscle tissue,this method will set the foundation to successful construction of three-dimensional model of muscle tissue in vitro.Two parts of our research.Part one:Biocompatibility of Sylgard 184 coated with different matrix materials and C2C12 cellsObjective:An attempt was undertaken to acquire the ideal matrix material that can enhance the compatibility between silicone rubber elastomer(Sylgard 184) and murine C2C12 cells.Methods:Two-component of sylgard184 admixture to the ratio of 10:1 then mixed into 4 holes of 6 Orifice,solidification at room temperature,and the remaining 2 holes without coating were taken as blank control(group A).The surfaces of 4 holes were coated by collagen typeâ… ,laminin and poly-lisine,respectively as group B,C and D,and the hole of uncoating as group E,every group has six samples.Each group C2C12 Cells morphology was observed by the inverted microscope.Flow cytometry (FCM) was used to detect the cell generation cycle of C2C12 cells after enrichment culture 48h.RT-PCR was used to test MyoD,Myogenin mRNA expression of proliferation or differentiation cultured for 48 h C2C12 cells respectively that growed on coated or uncoated sylgard 184 surfaces.Results:Material Sylgard 184 has cytotoxicities as vaccination C2C12 cells in group E all float deadly in 24h and most cells in group D become dead,only a small number of adherent cells are survival.While the toxicity of Sylagard 184 in group B and C are reduced significantly after materials are coated and the compatibility of C2C12 cells' surface are also enhanced.Besides,the percentage of cells in the synthesis phase,the quantity of MyoD in proliferating phase,and Myogenin differentiation gene mRNA expression in group C are significantly higher than that in group A and B(P＜0.05).Conclusions:The data suggests that Sylgard 184 modified by laminin is superior to that modified by other biomaterials,in terms of which can enhance the proliferation and differentiation of cultured C2C12 cells in vitro. Part two Construction and detection of highly organized 3dimensional muscle tissue in vitroObjective:To cultivate C2C12 cells on the modified mold-grooves of Sylgard 184 and induce C2C12 cells differentiation to highly organized 3-dimensional muscle tissue,then detect the growth and functional properties of differentiation myotube.Methods:Mix bicomponents Sylgard 184 by the ratio of 10:1 uniformly and poured the mixture into 6-well plates.After curing by standing at room temperature(The width of each mold groove is 0.8mm,depth is 0.15mm,length can adjust by requiring),carve grooves at the central basal Sylgard 184 and indentate in the groove at the bottom of the mold.After washing grooves with Hank's fluid,pave matrigel matrix and collagen (mixing ratio 1:6) mixture evenly at the bottom of grooves and add 0.3ml 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 C2C12 cell suspension whose density is 1.0 -1.2×10⁵ /ml,remove the entire medium when there are 80%cells proliferations,then add 2%horse serum containing DMEM/F12 and differentiation cultivate 7d,the inverted microscope to observe X-ray film morphology and continue to divide after the 21d,the construction of skeletal muscle tissue was detected by immunofluorescence assay muscle-specific protein expression,RT-PCR was used to detect Myogenin,Desmin mRNAs expression,while the use of electron microscopy to watch myotubes' morphologies and inter-connections.Results:C2C12 cells were differentiation cultivated in the mold-grooves of Sylgard 184 for 7 days,polarity-differentiation and myotube fusion were observed under the inverted microscope.After 21 days,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 was 0.15mm. Detect Myogenin,Desmin mRNAs of positive expression in constructed skeletal muscle tissue by RT-PCR,and there is differentiation-specific gene protein Myogenin and Desmin myoblast-specific protein expression by immunofiuorescence assay and DAPI nuclear staining.Results show that Myogenin expressed in differentiated myotubes and there is a large number of Myogenin in nucleis.While the positive expression of fluorescent protein showed that has the big differentiation potential. 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 Sylgard 184 is able to guide the differentiation of C2C12 cells and promotes them to form parallel multinucleated myotubes.Myotubes could overlap with each other and form three-dimensional skeletal muscle tissue.