Collagen/agarose/chitosan For The Construction Of Epidermal Substitutes In Vitro

Tissue engineered skin builds skin substitutes by culturing cells in vitro and biological scaffolds,among which epidermal substitutes are an important part of tissue engineered skin.When a single biological material is used as a 3D scaffold for cell growth in vitro and is used to construct an epidermal substitute in vitro with seed cells,there are defects such as poor mechanical properties and poor biocompatibility.The use of mixed biomaterials for cell culture and in vitro construction of epidermal substitutes can compensate for the defects of different biomaterials,which is more conducive to the construction of in vitro epidermal substitutes.In this paper,for the purpose of constructing epidermal substitutes in vitro,three different biological materials,agarose,chitosan and collagen,were mixed to prepare a 3D environment for cell growth in vitro.The constructed biomaterial scaffold was evaluated to study its feasibility as an epidermal substitute.The specific research contents are as follows:(1)The final concentration of 4 wt% agarose-chitosan scaffold was prepared by freeze-drying technology,and the concentration ratio of agarose to chitosan was 1:3,2:2,and3:1.By testing the density,porosity,swelling rate,degradation rate,biocompatibility and other aspects of the agarose-chitosan scaffold,the feasibility of the agarose-chitosan scaffold as an in vitro construction of epidermal replacement was evaluated.(2)The agarose-chitosan scaffold is improved,and a layer of collagen is embedded on the surface of the agarose-chitosan scaffold.The physical properties,mechanical properties,and biocompatibility of the collagen-coated agarose-chitosan scaffold and the collagen-uncoated agarose-chitosan scaffold were compared.The two scaffolds did not show significant differences in physical properties such as density,porosity,swelling rate,and degradation rate.Immortalized keratinocytes(Hacat)were seeded on collagen-coated agarose-chitosan scaffolds and non-collagen-coated agarose-chitosan scaffolds.On the third and fifth days of culture,the scaffolds Live/dead staining and immunofluorescence staining are performed on the cells.The two scaffolds have different promotion effects on cell proliferation and differentiation.Collagen-coated agarose-chitosan scaffold can promote cell proliferation and differentiation and is more suitable for constructing epidermal substitutes in vitro.(3)Collagen occupies a large proportion in human skin.In order to construct an epidermal substitute closer to human skin,collagen and agarose are mixed in different proportions to prepare collagen-agarose scaffolds.The physical properties,mechanical properties,and biocompatibility of different ratios of collagen-agarose scaffolds were evaluated.The higher the agarose content,the better the mechanical properties of the collagen-agarose scaffold,but the biocompatibility decreases.When the agarose content is35%,the collagen-agarose scaffold can take into account both mechanical properties and biocompatibility.At this time,the collagen-agarose scaffold is most suitable for constructing epidermal substitutes in vitro.