Study Of Biological Characteristics Of Myogenic Stem Cells Of Spatial Culture In Vitro

Skeletal muscle tissue engineering has great potential in improving the quality of transplanted cells and increasing survival rate. At present, skeletal muscle tissue engineering faces with enormous challenges in three-dimensional culture and tissue building. It is difficult to repair structure loss and dysfunction by transplanting myogenic stem cells in vivo. Because natural organization and plenitudinous regeneration capacity are needed, the vitality is poor and the compatibility is insufficient when the graft is transplanted into the host. Many researchers have done much work in skeletal muscle tissue engineering to structure functional muscle tissue. For example, Dennis and Kosnik have explored three-dimensional self-assembly model of skeletal muscle. They cultured myogenic stem cells and mechanocyte together. Myotubes were formed by myoblasts which were wraped with extracellular matrix secreted by mechanocyte, then generated continued-excitement and contraction when they were stimulated with transverse current. Baches' seeded myogenic stem cells to fibrin scaffolds and then cultured them to get polar multinucleated myotubes. Sxena seeded myogenic stem cells to polyglycolic acid meshs and then transplanted the complexes to the body. Finally he obtained vascularized-skeletal muscle tissue. Although foreign researches have involved in three-dimensional model of skeletal muscle tissue, all of they are in primary stage. Moreover there are no articles about three-dimensional building skeletal muscle in domestic at present. In view of current situation, our research group has done some preliminary exploration in this area. It is well known that skeletal muscle tissue engineering consists of three elements, namely, stem cells, scaffold and growth factors. It is the main areas of tissue engineering that the interaction between cells and matrix material research in order to promote cell adhesion and growth on host material. The key of the construction of three-dimensional skeletal muscle is whether we should obtain polar and parallel myotubes in vitro. The characteristic of homogeneous alignment of muscle fibre in mature muscle is the morphological basis of contractile function of muscular tissue. Our used C2C12 cells and three-dimensional scaffold gel of silicone elastomer co-culture and gain polar myotubes. Sylgard 184 is a kind of silicone rubber elastic material. This material has smooth surface and a good capability of elasticity so that they can be used in the detections of cell locomotion and the traction force.There was a preliminary exploration of three-dimensional culture in vitro on early work. Screened-sylgard 184 modification packages was the best support matrix material to make muscle cells that have a very good compatibility with matrix materials, and controlled the feature of the cell proliferation and differentiation in the material surface. Preliminary work is to explore the possibility of culturing tube with directional differentiation of the muscle in vitro three-dimensional culture. On this basis, we will further improve the technology of skeletal muscle in vitro three-dimensional structure and skillfully master the training method about polarity parallel to the muscle, then improve the Sylgard 184 casting groove, through the groove on the cast of C2C12 cells differentiation induced polarity, and use the mixture of extracellular matrix Matrigel and collagen in Sylgard 184 to optimize the mold cavity surface modification. After the work of inducing polarity, promoting differentiation, promoting extended of the modified groove and induced to differentiate, we obtain high density, polar differentiation with contractile protein expression and myogenic transcription factors. Its structure is more close to their physiological state. And the effect of myogenic stem cells growth and differentiation in Sylgard 184 three-dimensional matrix is detected. In order to better build muscle tissue in vitro, we tried to apply biological scaffold materials combined with PGA and C2C12 cells, and to explore the compatibility and feasibility in vitro. Compared with the morphology and function difference in three-dimensional and plane cultured C2C12 cells, then provide theoretical and technical support for the next muscle tissue three-dimensional stress and tension building. The components relatively sample about differentiation of myoblasts and myotubes in vitro could avoid the interference of other factors. This is a good model to study the muscle cells and muscle tissue and their functional activity. And then it helps us to understand some of the basic principles in cell development. This model is expected to be able to replace skeletal muscle in vitro, load the corresponding processing factors and simulate pathology conditions for studying the various myopathies.Three parts of the research:Part one:Sylgard 184 three-dimensional matrix affect the growth and differentiation of myogenic stem cellsObjective:To obtain the polar myotubules through seeding C2C12 cells to special casting surface of Sylgard 184, and detect the effects of growth and differentiation of myogenic stem cells on the three-dimensional matrix of silicone elastomer material.Methods:Mixed evenly Sylgard 184 with 10:1 ratio of two-component and poured the mixture into 6-well plate. To be impressed mold on the surface of semi-curing Sylgard 184, marked it length 25mm, width 0.8mm, depth 1mm. They were standby application after coated with the mixture of I collagen and matrigel matrix. To inoculate C2C12 cells suspension with density of 1×106/ml, when the cells fusion about 80% using 2% horse serum to do differentiation culture. Observe the structure and differentiation state of myotubule by inverted microscope, HE staining, RT-PCR and immunofluorescence.Results:After 10 day's induction, C2C12 cells on Sylgard 184 slots fused into polynuclear and polar myotubes. At the culturing time of 20 d, the myotubes became matured, with extended diameter, parallel distribution and the further confluence. This constructed muscle tissue shows automatic contractility, HE staining showed there are a large number of nuclei within parallel myotubes. PCR detection and immunofluorescence assay detection shows that specific marker proteins positive express. The results proved that it had muscle characteristics and contractibility of the muscle tissue analogues in vitro. Conclusions:The modified mold-grooves of Sylgard 184 were able to guide the differentiation of C2C12 cells and promote them to form parallel multinucleated myotubes. Experiments show that the modification of Sylgard 184 three-dimensional scaffold provided a place of boarding, proliferation and differentiation for myogenic stem cells. It has good biocompatibility and can enhance cell adhesion and proliferation, lead multi-core muscle control polar growth, promote of muscle-like differentiation, guide tissue regeneration. The obtain material had biological characteristics and structure of muscle. Provide the experimental basis and technical support for stress loading of muscle tissue, as well the model of skeletal muscle diseases.Part two:Evaluation of the biocompatibility of PGA fibers with skeletal muscle stem cells in vitroObjective:To evaluate the biocompatibility of PGA with C2C12 cells and the ability of seeding methods for developing tissue engineered muscle tissue analogues in vitro.Methods:Loose PGA fibers were fixed to the surface of the coverslip to ensure the fibers were parallel and have an appropriate tension. The length of PGA fibers bundle was 3cm and the diameter was about 0.2 cm. They were sterilized by dipping in iodine and ethanol, washed with 0.1% PBS. Subsequently, ultraviolet radiation for 30 min after coated PGA with type I collagen and Matrigel Matrix. Then it was pre-cultured 3 days in 37℃5% CO2 incubator with containing 10% FBS in DMEM/F12 medium. C2C12 cells with concentrated suspension were seeded on PGA fibers bundle and co-cultured, differentiation. Observe cell attachment and tissue development on the PGA scaffolds by light microscopy and scanning electron microscope.Results:Under the inverted microscope, C2C12 cells on the scaffold were in well shape, and relatively few dead cell, it shown round spindle cells adhere on the surface and the gap of the PGA fibers. The cells extended to the poles along the longitudinal axis of PGA fibers and had good compatibility with the scaffold. After 7 day's differentiation, the muscle tissue analogue was observed under the SEM. There were many myoblasts adhering on the surface of PGA fibers and had strong secretion of extracellular matrix and the myotubes fused a membranous structure along the direction of parallel PGA. It has strong secretion of extracellular matrix. It survived well but differentiated unevenly and they were not uniform length and diameter. PGA has excellent biocompatibility with C2C12 cells.Conclusions:PGA is the most widely used as the tissue engineering scaffold material and was favored by tissue engineering researchers of all ages for its good biocompatibility and degradation characteristics in vivo. As the PGA fibers arranged in parallel bundles and made the cells suspension permeated well, it is beneficial to growth and polarity differentiation of skeletal muscle stem cells. The SEM studies had preliminary confirmed that myoblasts have good biocompatibility and differentiation potential on the PGA fibers surface, and indicated that the feasibility of PGA fibers as the scaffold material can be used for building muscle tissue in vitro.Part three:Comparative study of spatial and monolayer culture of myogenic stem cells in vitroObjective:To compare morphologic and functional features of C2C12 cells cultured on monolayer or Sylgard 184.Methods:C2C12 cells were cultured on the dishes or the Sylgard 184 slots modified by type I collagen and Matrigel Matrix respectively.2% horse and confluction about 80%. The morphologic serum was added into DMEM/F12 for priming the cell differentiation after the cell proliferating and functional features were explored, adopting the inverted microscope, RT-PCR, and immunofluorescence.Results:After 5 day's induction, C2C12 cells on Sylgard 184 slots fused into polynuclear and polar myotubes. At the culturing time of 17d, the myotubes became matured, with extended diameter, parallel distribution and the further confluence. This constructed muscle tissue showed automatic contractility, with the thickness of about 0.3mm. Under the scanning electron microscopy, myotubes closely arranged and overlapped with each other. Compared with that of spatial culture, the differentiated myotubes of monolayer culture was tiny, arranged more randomly. The results of RT-PCR proved that, spatial culture prompted to the expression of MyoD and Myogenin mRNA, compared to monolayer cultures, as well the protein levels of Desmin, F-actin, MHC, and nAChR.Conclusions:Compared with the traditional monolayer culture, three-dimensional culture is preferential to the differentiation, paralleled arrangement and the expression of functional molecules of myotubes in vitro, which is a favorable model to explore the development and stress loading of muscle tissue, as well the principals of skeletal muscle diseases.