Mechanically Inducted Three-Dimensional Hydrogel Fiber Alignment and its Application in Skeletal Muscle Regeneration

Skeletal muscle accounts for 40%~45%of the body weight and is composed of a large number of parallel arrangement of muscle fiber bundles through attaching to the skeleton by the tendon.And robust skeletal muscles provide people the ability of daily activities such as walk,lift and so on.Muscle fibers are generated by multinucleated myotubes which form from the fusion of myoblasts with polarity development,and the synergistic effect between muscle fibers and spontaneous contraction provides support for the movement of the bodies and limbs.However,because of common clinical genetic defect,trauma,tumor,primary myopathy there are so many people cannot exercise like normal people.So it is an urgent task to treat these diseases.Traditional treatment methods are mainly through acupuncture,rehabilitation,drug treatment,etc.But these methods have long treatment times,poor efficacy,and high costs.Recently,the clinical researchers find that these diseases have similar pathological characteristics including structural damage,loss of skeletal muscle function,apoptosis and degradation of muscle fibers Therefore,there is academic and clinical value to study the repair and regeneration of skeletal muscle.Nowadays,tissue engineering provides a new idea for repairing damaged muscle tissue.Tissue engineering uses biological materials as a carrier to regulate the structural properties of materials.By applying biological/chemical/physical stimuli to mimic the microenvironment of the living body.And then construct a living tissue and transplant it into the body for wound repair.With the development of tissue engineering,we regulate the micro-structure of the cell matrix and applying mechanical stimulation to reconstruct a functional muscle microtissue in vitro.Such fabrication and mechanical loading strategy provide an easily adaptable platform to create functional muscle tissue constructs.And the mature muscle microtissues provide a useful tool for the clinical application to treat muscle injury.In this work,we regulate ECM microstructure and apply mechanical stimulation to construct three-dimensional muscle cell mechanical microenvironment.We first regulate the collagen fiber orientation by utilizing the characteristics of collagen gelation,which is a slow process and often need half an hour even more.Before gelation,we fabricate a PDMS mould with rectangle chamber and apply a strain along the long axis to a certain extent utilizing its elasticity.Then we pour collagen solution into the chamber and release the force a few minutes later and gelatinize.After gelation,we apply stretching on the end of the rectangle collagen hydrogel to promote further alignment.We adjust the strain loading time and the strain level to form different groups.Determining the optimal experimental conditions for myotube formation by counting the length and area ofthe myotube and immunofluorescence about relevant proteins.Later,we use western blot and RT-PCR to prove our result.It was found that the application of pre-strain and tensile stimulation can promote the alignment of collagen fibers.Results show that strain loading time and strain extent can regulate the morphology and function of myotubes.The muscle mechanical microenvironment we constructed promotes the formation and activity of polar multinuclear myotubes and builds mature muscle tissue,and determines the related molecular pathways and their transmission mechanisms during the mechanical conduction process.