Objective To investigate the effect of hydroxyapatite/zirconia (HA/ZrO2) scaffold by three-dimensional printing compounded with vascular endothelial growth factor (VEGF) 165 calcium alginate microsphere slow-release system on repairing femoral shaft defects in dogs.Methods The HA/ZrO2 artificial prosthesis was prepared by three-dimensional printing,and the co-culture system of slow-release system of composite VEGF 165 calcium alginate microspheres was constructed.Sixteen beagle dogs were divided into four groups according to the extent of femoral shaft interception,with four dogs in each group.Group A:no biomaterials were implanted into the middle femur of dogs after 15 mm of femur interception as blank control group;Group B:HA/ZrO2 scaffolds composite with VEGF165 calcium alginate microspheres were implanted into the middle femur of dogs after 15 mm of femur interception;Group C:the same method as Group B was adopted after 25 mm of femur interception;Group D:the same method as Group B was adopted after 35 mm of femur interception.General examination and X-ray imaging observation were taken after operation.The ability of new HA/ZrO2 gradient biocomposites to repair bone defects was evaluated by micro CT scanning,biomechanical testing,ink staining and toluidine blue staining 12 weeks after operation.Results The drug loading capacity of calcium alginate microspheres reached (23.6 ± 2.9) ng/mg,and the entrapment efficiency reached (62.4 ± 3.6) ％,showing a slow rate of release.Gross examination showed surgical incision was healed in all four groups.Postoperative X-ray imaging of experimental animals showed that nonunion was formed in Group A over time;in Group B,the artificial prosthesis was gradually filled with new bone and the boundary was blurred;in Group C,the early reaction was slower than that in Group A,and the callus passed continuously 12 weeks after operation;in Group D,new bone formation was slow,only surrounding the broken end.At 12 weeks after operation,the neonatal bone mass was (238.6 ± 19.1)mm3 in Group B,(223.3 ± 13.4) mm3 in Group C,and (110.8 ± 6.5) mm3 in Group D.There were significant differences among the three groups (P ＜ 0.05),but no significant difference was found between Group B and Group C (P ＞ 0.05).The results limit compression test at 12 weeks after operation showed no significant differences among Groups B [(49.7 ± 2.3) MPa],C [(49.81 ± 2.4) MPa] and D [(46.9 ± 3.6) MPa](P ＞ 0.05).At 12 weeks after operation,the histological sections showed that the blood vessels in Groups B and C were thickened,with obvious branches,and the surrounding new bone increased.During the period,the blood vessels were filled with vascular network.There were no obvious differences in the number and shape of blood vessels between groups.However,Group B had more new bones and blood vessel networks.New bone and small vascular networks were seen in Group D.Conclusion The hydroxyapatite/zirconia scaffold by three-dimensional printing compounded with vascular endothelial growth factor 165 calcium alginate microsphere slow-release system can repair dogs' femoral bone defect within 35 mm.