Study On Preparation Of Conductive And Elastic Cryogel And Its Effect On The Repair Of Damaged Skeletal Muscle

Part One:Exploring the in vitro cytocompatibility of dopamine-conducted cryogel and promoting the proliferation of C2C12 cellsObjective:In this study,the mussle-inspired polypyrrole(Ppy)-methacrylated gelatin(GelMA)cryogel(Dopamine-based polypyrrole-methacrylated gelatin cryogel,Ppy-GelMA)was investigated as a scaffold to enhance the proliferation of C2C 12 and the potential scaffold of the engineered skeletal muscle tissue.Methods:The structure of the material was observed by scanning electron microscopy.The growth of cells was confirmed by scanning electron microscopy after C2C12 cells were seeded on the DOPA-base Ppy-GelMA.The viability of the cells was determined by live/dead staining.CCK-8 Tests,Ki-67 immunofluorescence staining were used to study the effect of materials on promoting cell proliferation.Results:The Ppy-GelMA had the uniform and interconnected porous structure.Interestingly,myoblasts in Ppy-GelMA were oriented aligned.Both the GelMA and the Ppy-GelMA were suitable for the attachment and spreading of C2C12 myoblasts,while the cell viability in Ppy-GelMA hcryogel was significantly higher than that in GelMA.Further,the Ki67 positive myoblasts rate in Ppy-GelMA was sighnificantly greater than that in GelMA.Conclusion:This Ppy-GelM A can enhance the proliferation of the myoblasts.Part Two:Study the effect of the dopamine conductive cryogel material in gastrocnemius muscle injury mice in vivoObjective:Myoblasts-loaded Ppy-GelMA was implanted into the gastrocnemius of the skeletal muscle injury mouse models and invested the regeneration of the injury muscle.Methods:The skeletal muscle injury model was established by injection of cardiotoxin(CTX)into the gastrocnemius muscle of mice.There are four groups are established:PBS group,CTX injury group,GelMA+cell group,and Ppy-GelMA group.After the C2C12 cells were seeded onto the GelMA and Ppy-GelMA,they were implanted into the gastrocnemius muscle of mice.The gastrocnemius muscle contraction force was measured in vivo at the first week and the third week.The mice were then sacrificed and the gastrocnemius of mice were weighed to obtain the Wet weight.Then muscle freeing and make into frozen setion for HE staining,or 74/80 immunofluorescence staining and observations by optical microscope:i)necrosis and regeneration of muscle fibers,ii)exudation infiltration of macrophages RESULTS;At 1 week,the contractility and wet weight of gastrocnemius were measured.Except for PBS group,the gastrocnemius muscle contractility of Ppy-GelMA group was the highest,and there was a statistically significant difference compared with the injury group.At 3 weeks,the gastrocnemius muscle contractility and muscle wet weight of each CTX injury group were increased.The gastrocnemius muscle contractility and muscle wet weight of Ppy-GelMA group were still the highest,indicating that the skeletal muscle function recovery was the best.After freezing sectioning,HE staining showed that the inflammatory infiltrates were intense at 1 week between the injury model groups,whereas the CTX injury group had higher inflammatory infiltration at 1 week and 3 weeks than the material group.Ppy-GelMA Inflammation of the group was the lowest.According to the results of HE staining,the muscle fibers in the Ppy-GelMA group at 1 week and 3 weeks were the largest among the injury model groups.The results of immunofluorescence staining with F4/80 showed that at 1 week each model group had strong macrophage infiltration,and at 3 weeks,the macrophage infiltration in each group decreased significantly,while Ppy-GelMA.The group had the lowest degree of macrophage infiltration.Results:At 1 week,the gastrocnemius muscle contractility and wet weight of muscles were measured.Except for PBS group,the gastrocnemius muscle contractility of Ppy-GelMA group was the highest,and there was a statistically significant difference compared with the injury group.At 3 weeks,the gastrocnemius muscle contractility and muscle wet weight of each injury group were increased.The gastrocnemius muscle contractility and muscle wet weight of Ppy-GelMA group were still the highest,indicating that the skeletal muscle function recovery was the best.After cryosectioning,hematoxylin and eosin staining showed that the inflammatory infiltrates were intense at 1 week between the injury model groupsConclusion:Implanting the conductive cryogel into the gastrocnemius of the injury mice models can promote the recovery of the muscle strength of the damaged skeletal muscle,reduce the local inflammatory infiltration,and promote the regeneration and repair of muscle fibers.Therefore,the conductive cryogel is a promising skeletal muscle tissue engineering scaffold.