Study On The Properties Of Konjac Glucomannan–Fibrin Hydrogels And Their Application In Cell Scaffolds

With the development of cell biology and tissue engineering,cultured meat has become a new way of the production of meat.The research in cell-based cultured meat focuses on the construction of cell scaffolds.The ideal cell scaffolds should have good biocompatibility and degradation characteristics,and the mechanical properties of scaffolds could affect the differentiation of stem cells growing on the scaffolds.Polysaccharides and proteins are two of the most important macromolecular polymers in the food system,which are very suitable.for the preparation of cell scaffolds.Konjac glucomannan(KGM)is a natural water-soluble polysaccharide extracted from Konjac,which can be deacetylated to form thermally irreversible hydrogels under alkali conditions.However,KGM hydrogels have poor water-holding capacity and biocompatibility,which limit the application of KGM in cell scaffolds.Therefore,KGMfibrin(FN)composite hydrogels of different mechanical properties were prepared in this study to support the myogenic and osteoblastic differentiation of the ectodermal mesenchymal stem cells(EMSCs).The main results of this study were as follows:(1)Polysaccharide-protein composite hydrogels with different ratios of KGM and FN were prepared,and the mechanical properties,rheological properties,water-holding capacity,degradation rate,microstructure,and biocompatibility were measured.The results indicated that the gel strength,degradation rate,and water-holding capacity of the composite hydrogels were better than those of the single gels.The KGM-FN hydrogels(1.6% KGM,0.4% FN)had a Yong’s modulus of 24.94 k Pa ± 0.08 k Pa,which were beneficial to the myogenic differentiation.Composite hydrogels(0.8% KGM,1.2% FN)had a high Young’s modulus of48.67 k Pa ± 2.78 k Pa,which was conducive to the osteogenic differentiation of the stem cells.The KGM-FN composite hydrogel is a shear-thinned non-Newtonian fluid.The major intermolecular forces between KGM and FN were hydrogen bond interaction,which helps to form a more suitable gel structure.KGM-FN composite hydrogels have uniform pores and excellent biocompatibility,which were conducive to the proliferation of EMSCs without affecting cell morphology.(2)The effects of KGM-FN composite hydrogels with low Young’s modulus on the myogenic differentiation of EMSCs were further investigated to explore their usage in the preparation of cell-cultured meat.The results showed that compared with composite hydrogels with high Young’s modulus,the soft KGM-FN composite hydrogels(0.8% KGM,1.2% FN)could upregulate the expression of myogenic-related genes including Desmin,Myf5,Myogenin,and Myod1,and promote myogenic differentiation of EMSCs.These promoting effects may be related to the inhibition of Rho/Rock pathway.KGM-FN composite hydrogels were used to prepare cell-cultured meat,and the texture characteristics and main nutrients of cell-cultured meat were similar to those of animal meat,and the content of dry protein and dry carbohydrate were higher than traditional meat.(3)The effects of KGM-FN composite hydrogels with high Young’s modulus on the osteogenic differentiation of EMSCs were investigated in vitro and in vivo.The results showed that composite hydrogels with the high Young’s modulus(1.6% KGM and 0.4% FN)could increase the activity of alkaline phosphatase,promote the formation of calcium nodules,and up-regulate the expression of osteogenesis-related genes OCN,OPN,Smad1,Smad5,and RUNX2,thus were conducive to the osteogenic differentiation of EMSCs.EMSCs on the surface of the composite hydrogels sensed the mechanical stimulation of extracellular matrix through the cell membrane integrin β1,and activated the FAK/ERK signaling pathway,thereby up-regulating the expression of osteogenesis-related genes.The rat alveolar bone defect model was established to verify the effect of the composite hydrogels on promoting the osteogenic differentiation of EMSCs in vivo.It was found that compared with the untreated model group,the composite hydrogels loaded with EMSCs could up-regulate the expression of osteogenicrelated genes,promote the formation and mineralization of bone matrix,and promote the osteogenic differentiation of stem cells in vivo,which has a broad application prospect in related fields.