Preparation And Preliminary Study Of Polysaccharide Hydrogel As Scaffold For Muscle Stem Cell Culture

The ability of skeletal muscle to repair and regenerate is greatly affected or even lost when skeletal muscle is severely damaged.Skeletal muscle tissue engineering technology is a promising strategy for promoting skeletal muscle regeneration.In order to solve the contradiction between the growing population and the increasing demand for animal protein,in vitro cell culture meat is increasingly developed,which was the main use of skeletal muscle tissue engineering technology.Natural as a scaffold for tissue engineering,they have great advantages in supporting cell growth.However,natural polymer hydrogels have the disadvantages of low mechanical strength and poor stability.Based on this,in this paper,Ca CO₃ was used as the cross-linked calcium source of sodium alginate（SA）to prepare a physical cross-linked double network CS/SA/Ca²⁺hydrogel scaffold with chitosan（CS）and SA crosslinking network as the main network,aiming to prepare an ideal cell culture scaffold for skeletal muscle tissue engineering by optimizing the combination of materials.（1）By preparing CS/SA/Ca²⁺hydrogel scaffolds with different CS/SA/Ca²⁺molar ratios,the effects of CS/SA/Ca²⁺molar ratios on the physical and chemical properties of hydrogels such as gel uniformity,internal microstructure,and mechanical strength were studied.Scanning electron microscopy（SEM）results showed that CS/SA/Ca²⁺hydrogel scaffolds had good three-dimensional morphology and porous structure.Energy dispersive spectrometer（EDS）results showed that CS,SA,and Ca CO₃ materials were uniformly distributed and efficiently gelled in the hydrogel.When the molar ratio of CS/SA/Ca²⁺was 1:1.2:0.24（H-1.2group）,the pore size of the CS/SA/Ca²⁺hydrogel scaffold was 104.06±2.64μm,which was conducive to cell adhesion and migration.The compression modulus is 40.00±2.52 k Pa,which is close to the natural skeletal muscle.The viability of cells cultured in the extracts of CS/SA/Ca²⁺hydrogel scaffolds in each group was more than 90%,indicating that CS/SA/Ca²⁺hydrogel scaffold had good biocompatibility.（2）In vitro cell culture evaluation of CS/SA/Ca²⁺hydrogel scaffolds were performed.SEM and confocal laser scanning microscopy（CLSM）showed that the activity and growth rate of C2C12 myoblasts on CS/SA/Ca²⁺hydrogel in the H-1.2 group were the highest.The H-1.2 group CS/SA/Ca²⁺hydrogel as a scaffold for the culture of commercial porcine muscle stem cells（PMu SCs）could provide a space conducive to the adhesion,growth,and proliferation of stem cells.Further,primary muscle stem cells（Mu SCs）were further isolated and extracted from the longissimus dorsi tissue of piglets,and Mu SCs with high purity were separated by surface labeled proteins CD31^-/CD45^-and CD56⁺/CD29⁺.Surface antigen markers Pax7 and Myo D were detected by cell immunofluorescence technology,and both of them were positively expressed in the cells obtained,indicating that Mu SCs with high purity and strong activity were isolated and extracted.The results of myosin heavy chain My HC immunofluorescence were positive after induction differentiation of the extracted Mu SCs,indicating that the Mu SCs had myogenic differentiation ability.The successfully extracted Mu SCs were inoculated on CS/SA/Ca²⁺hydrogel scaffolds for in vitro cell culture experiments.Mu SCs also showed better adhesion,growth,and proliferation effects on CS/SA/Ca²⁺hydrogel scaffolds in the H-1.2 group,indicating that adjusting the CS/SA/Ca²⁺molar ratio could improve the adhesion and growth environment of cells on CS/SA/Ca²⁺hydrogel scaffolds.In this study,the physically cross-linked dual-network CS/SA/Ca²⁺hydrogel was successfully prepared.The hydrogel has good printability,and the CS/SA/Ca²⁺hydrogel with a certain CS/SA/Ca²⁺molar ratio is expected to become one of the stem cell culture scaffolds for skeletal muscle tissue engineering.