Abstract: BACKGROUND: Collagen/chitosan scaffolds need to be cross-linked to achieve corresponding mechanical properties. Studies have shown that the regulation of crosslinking agent concentration can control the physical and chemical properties of collagen/chitosan scaffolds. 
OBJECTIVE: To explore the effect of genipin concentration on physical and chemical properties of collagen/chitosan scaffolds, and manufacture tissue engineering scaffolds with adjustable physical and chemical properties. 
METHODS: Collagen and chitosan were separately dissolved in weak acid and then mixed as printing ink. The collagen/chitosa scaffolds were printed using biological 3D printer at low-temperature, then lyophilized and neutralized, and crosslinked with 1, 3 and 5 mmol/L genipin. The apparent structural stability, tensile strength, swelling performance, degradation performance and biocompatibility of scaffolds of each group were detected. 
RESULTS AND CONCLUSION: (1) After the scaffold was soaked in PBS for 3 days, compared with the uncrosslinked freeze-dried scaffold, the surface of the collagen scaffold maintained regular pore structure after crosslinking, but the scaffold showed obvious deformation. The surface structure of the collagen/chitosan scaffold was regular after crosslinking. The collagen/chitosan scaffold cross-linked with only 1 mmol/L genipin had slight deformation. (2) With the increased concentration of genipin, the mechanical properties of scaffolds of each group increased, and the mechanical properties of collagen/chitosan scaffolds at the corresponding crosslinking concentration were better than those of collagen scaffolds. (3) With the increased concentration of genipin, the swelling rate of the collagen scaffold decreased, and the swelling rate of the collagen/chitosan scaffold did not remarkably change. (4) After soaking in collagenase solution, the collagen scaffolds crosslinked with different concentrations of genipin were totally degraded within 1 hour. The degradation rate of collagen/chitosan scaffold decreased with the increased concentration of genipin, showing a trend of rapid first and then smooth. (5) After inoculating bone marrow mesenchymal stem cells on the crosslinked scaffolds for 3 days, the number of cells on the 1 and 3 mmol/L genipin crosslinked collagen/chitosan scaffold (or collagen scaffold) was significantly more than that on the 5 mmol/L genipin crosslinked collagen/chitosan scaffold (P < 0.05). (6) Results have suggested that genipin can adjust the physical and chemical properties of collagen/chitosan scaffolds within a certain extent. Among them, collagen/chitosan scaffold crosslinked with 3 mmol/L genipin has good mechanical properties, resistance to enzymatic hydrolysis and biocompatibility.

Key words: crosslinking, collagen, chitosan, low-temperature 3D printing, genipin, tissue engineering, scaffold, cell viability