Research Of Uniaxial Stretching-induced Formation And Actuation Characteristics Of Skeletal Muscle Based On GelMA Microfibers

Bio-hybrid actuators,powered by contractible cells or tissues,have become the main source of power for bio-inspired robots due to their intrinsic safety,high energy efficiency,low energy consumption,high sensitivity,and self-repair capabilities.Among the common living contractible biological materials,the tissue engineered skeletal muscle has a wide source of myogenic cells,low tissue engineering difficulty,high degree of controllability,high power-to-weight ratio,and self-healing capabilities,making it the most potential source of power for bio-hybrid actuators.As a common fiber-shaped tissue within human body,the structure of skeletal muscle has an important impact on its functional integrity and application scenarios of skeletal muscle-based actuators,so it is necessary to replicate its microfiber-shaped structure in vitro.Nevertheless,existing microfiber processing methods are not suitable for the construction of fiber-shaped cell-derived constructs due to their extreme processing conditions,detrimental to cells due to multi-step curing or coagulant agent exposure.Natural skeletal muscle is always under mechanical field,which plays a key role in skeletal muscle formation.However,the systematic study of skeletal muscle formation in vitro induced by mechanical stimulation remain challenges in operation system controlling,space occupation or the risk of contamination.Besides,the limited actuation ability and lifetime of tissue engineered skeletal muscle have brought many challenges to its application in bio-hybrid actuation.For the needs of bio-hybrid actuation based on engineered skeletal muscle,this paper fabricated densely packed,long-lifetime and high-performance skeletal muscle based on gelatin-methacryloyl(GelMA)microfibers.The main research contents and results are list as follows:1.Fabrication of GelMA microfibers imitating skeletal muscle extracellular matrix.The silicone-tube-based coagulant bath free method is proposed for GelMA microfibers fabrication.Using this methods,tens of centimeter long homogeneous cell-laden microfibers can be obtained under single UV exposure.This method is widely applicable due to its easy-operation,low material limitations and low cost.By changing the exposure time,we generate microfibers with structure and stiffness similar to native skeletal muscle.Besides,the soft polymerization condition and the excellent physical properties of GelMA are beneficial for nutrient diffusion and high cell viability maintaining during culture,which makes it has high potential in skeletal muscle tissue engineering.2.Systematic investigation of uniaxial stretching-induced microfiber-shaped skeletal muscle formation.We designed a pillar-well array-based stretching device to apply different degree of uniaxial strain flexibly to regulate C2C12 cells behavior.This independent miniaturized stretching device is reusable,low-cost,low energy consumption,and highly flexible.Integrated with the culture system,it can provide a research platform for the systematic analysis of mechanical-biological response-With the loading of uniaxial strain,the cell spreading,elongation and alignment was effectively improved.As for the size,differentiation maturity of myotubes,they increase as well and reach a saturation level as strain ratio>35%.The above results indicate that uniaxial stretching can effectively enhance the formation and maturation of skeletal muscle.The actuation performance of microfiber-shaped skeletal muscle has excellent controllability,and as the strain increases,its actuation ability is significantly improved,and the actuation mode changes from torsion mode(strain<25%)to linear contraction(strain>25%).The mechanical response of myoblasts in microfibers and the systematic analysis of myotube formation have provided us with a deeper understanding of the role of mechanical stimulation at various stages of skeletal muscle formation.The influence of uniaxial stretching on the actuation ability and actuation mode of microfiber-shaped skeletal muscles makes bio-hybrid actuators can be customized more convenient,thereby broadening its application field.3.Fiber-shaped skeletal muscle actuation performance optimization research.In order to further improve the cell density of fiber-shaped skeletal muscle,uniaxial stretching and insulin-like growth factor-I(IGF-I)are introduced.With their combining effects,the skeletal muscle formation within microfibers is regulated,which obviously promoted the increase of myotube density and size.In addition,the decline of myotube contractility with culture time is delayed,which is beneficial to prolong their service lifetime.Then,electric training engages in skeletal muscle engineering as an effective methods to improve the actuation performance of microfiber-shaped skeletal muscle.The combination of uniaxial stretch,IGF-I and electric training highly facilitates myoblasts proliferation and skeletal muscle formation,thereby dense and actuation performance improved myotubes are obtained within microfibers-shaped skeletal muscle,with the contraction displacement is about 3.2 times of the original.The microfiber-shaped skeletal muscle with dense and actuation performance improved myotubes,have great potential to be biohybrid actuators.The innovations of this paper are as listed follows:The silicone-tube-based coagulant bath free method is proposed to fabricate mimic-skeletal muscle extracellular matrix constructs.Besides,a miniaturized,low energy consumption pillar-well array-based stretching device is designed for cell behavior,skeletal muscle formation regulation and actuation mode modulation.In addition,the combined effects of uniaxial stretch,IGF-I and electrical stimulation training is introduced,which makes the contraction performance of microfiber-shaped skeletal muscle greatly improved,and delays the decline of its function,making it suitable for the application in biological hybrid actuation.