Studies On Coaxial Printing Of Vascularized Tissue Construction Models Based On Double Crosslinked Gelatin-Alginate Hydrogels

Objective:The creation of vascularized organs can be used for organ transplants,drug screening and other areas.However,there are some bottlenecks in their in vitro construction,for example,the connectivity of blood vessels is generally difficult to control and the endothelialisation of the inner lumen of the vessels is insufficient.Natural polymeric hydrogels,such as gelatin and sodium alginate,are currently the most used matrix materials in three-dimensional（3D）cell cultures,with excellent biocompatibilities.However,there is a large gap between the mechanical properties of these hydrogel materials and human soft tissues,and their mechanical properties are often needed to be enhanced through modifications or crosslinking.3D bioprinting is a cutting-edge technology that can precisely construct complex tissues and organs to mimic their natural counterparts.It has unique advantages in the construction of small-diameter blood vessels.In this study,a double crosslinked hydrogel was prepared by crosslinking gelatin with oxidized pullulan polysaccharide and sodium alginate with Ca²⁺to enhance its structural stability.When the cell-laden hydrogel was printed as a bioink using a coaxial 3D bioprinter,a small-diameter vascular network with endothelialised inner surface was constructed.Methods:Oxidized pullulan polysaccharide was firstly selected as a crosslinking agent to crosslink gelatin,where oxidized pullulan polysaccharide was obtained by oxidation with sodium periodate.The content of aldehyde groups in the oxidized pullulan polysaccharide,the crosslinking degree and cytotoxicity of the gelatin hydrogels were determined by Na OH titration,ninhydrin method,CCK-8,as well as self-healing performance test.It was compared with the enzymatic degradation rate of microbial transglutaminase enzyme crosslinked hydrogels to assess the oxidized the crosslinking effect of pullulan polysaccharide on gelatin.Afterwards,the gelatin-alginate solution was double crosslinked using oxidized pullulan polysaccharide and Ca Cl₂,and the hydrogels with suitable concentration of oxidized pullulan polysaccharide crosslinked were selected by observation of water retention,degradation rate and pore structure.Then the double crosslinked hydrogels mixed with adipose stem cells/human umbilical vein endothelial cells were assessed as bioinks.A vascular network was obtained using a coaxial bioprinter and it was characterized via live/dead cell staining,DAPI staining,HE staining and scanning electron microscopy to detect the survival status of cells within the hydrogel after printing.Additionally,a comined stromal hydrogel containing human tongue squamous carcinoma cells was printed to construct cancer tissue models with embedded vascular networks,and followed by immunofluorescence,phalloidin staining and connectivity testing characterizations.Results:（1）Gelatin hydrogels with good biocompatibility and self-healing properties were successfully crosslinked by oxidized pullulan polysaccharide,and the double crosslinked hydrogels prepared with sodium alginate have better structural stability than Ca Cl₂ single crosslinked hydrogels.（2）The double crosslinked hydrogels possess good printing performance and biocompatibility.（3）A continuous and complex vascular network with an average outer diameter of 1 mm was obtained through coaxial 3D bioprinting.The vascular network had a good state of inside cells,clear inner and outer walls,a certain pore structure,and go-through innere cavities.Conclusion:The selected oxidized pullulan polysaccharide as the gelatin crosslinking agent,is non-toxic,non-cooler reactive and simple to prepare.The double crosslinked gelatin-alginate hydrogels are biocompatible,stable,and suitable for being used as bioinks in 3D bioprinting.Furthermore,a cancer tissue model containing complex vascular network was constructed using this coaxial 3D bioprinting technology,which has provided a new idea for in vitro construction of complex organ models.