Experimental Study On The Treatment Of Large Segment Bone Defects With HA/ZrO₂ Gradient Composites By Three-dimensional Printing With Light Curing

Objective: 1.To prepare a magnetic calcium alginate sustained-release system which encapsulates vascular endothelial growth factor 165（VEGF165）and verify the release efficiency of the sustained-release system.2.To prepare nano HA/ZrO₂ gradient composite scaffold was prepared by three-dimensional printing technology,and to construct the co-culture system of nano-HA / ZrO₂ gradient composite scaffold and calcium alginate sustained-release system.The co-culture system was implanted into the defect area of Beagle’s femur,and studied its repair ability of bone defect.Method: 1.The sodium alginate（Alg）solution with concentration of 12.5 mg/ml was mixed with calcium chloride（Ca Cl2）solution of 45.2 mg/ml,preparation of calcium alginate microspheres by wrapping VEGF165,then freeze-dried.Scanning electron microscopy was used to observe the surface and internal morphology of calcium alginate microspheres,and Enzyme linked immunosorbent assay（ELISA）was used to detect the drug loading,encapsulation efficiency and sustained release efficiency of calcium alginate microspheres.2.The performance of gradient composite scaffolds was evaluated by scanning electron microscopy（SEM）,X-ray diffraction（XRD）,Cell Counting Kit-8（CCK-8）and biomechanical tests.HA / ZrO₂ gradient composite scaffold was constructed and implanted into Beagle’s femur defect model.12 Beagles were randomly divided into 4groups,with 3 dogs in each group.Group A: after intercepting 20 mm middle femur,the gradient composite was not implanted as a blank control group;Group B: After interception of 20 mm,the HA/ZrO₂ gradient composite was implanted as a experimental group;Group C: After interception of 25 mm,the HA/ZrO₂ gradient composite was implanted as a experimental group;Group D: After interception of 30 mm,the HA/ZrO₂ gradient composite was implanted as a experimental group.After operation,the dogs were to observed the general and wound healing situation;Take X-ray imaging to observe on the day after operation,4 W,8 W and 12 W;The dogs were sacrificed at 12 W after operation,the specimens were scanned by Micro CT,then to reconstructe three-dimensional,detecte new bone mass and biomechanical properties.Evaluation of bone defect repair ability of the gradient composites of nano HA/ZrO₂ coated with calcium alginate sustained-release system by all the indexes.Results: 1.The encapsulation efficiency of calcium alginate microspheres which were prepared with 12.5mg / ml Alg solution and 45.2mg / ml Ca Cl2 solution was 72.5%±4.1%,n=3;The drug loading was 29.0±1.6pg/mg;The sustained release efficiency was 9.4±0.7% in 12 h,68.1±1.8% in 7d,82.5%±2.5% in 9d and 76.0±1.6% in 12 d.2.The nano HA/ZrO₂ gradient composite had regular appearance,stable structure,uniform pore size and distribution.SEM observation showed that the graded composite material surface had the shape of shingle,high density,regular pore structure and uniform surface structure under high magnification.The XRD analysis showed that ZrO₂ in the gradient composite material presents multiple peaks,and the peaks are steep and obvious,and the purity of zirconia powder is high.CCK-8 experiment showed that the composite material had no obvious cytotoxicity.The ultimate compressive strength was 13.4±0.9MPa.The dogs after operation were healed,no obvious wound swelling and fluid exudation.Postoperative X-ray imaging showed: Group A,with the passage of time,there was no obvious callus formation in the broken end of the two fracture,with the bending of the plate and the loosening of the screw;Group B,The interface between the scaffold of gradient composite material and the fracture end of the fracture was gradually blurred,and the continuous callus passed through the two fracture end.The stent was connected to the fracture end at 12 W after the operation;Group C: The fracture end and the gradient composite scaffold were gradually filled with the new bone,but a few scaffolds were broken.After 12 W,the continuous callus was shown to pass through the fracture;Group D: Although the growth of the new bone tissue was slow,the scaffold was connected with the fracture end after 12 w.Dogs were sacrificed at 12 W after operation,and the specimens were reconstructed and the new bone mass was analyzed by Micro CT.The fracture ends of group A were not healed,which belonged to the nonunion.Group B,group C and group D were all formed with new bone tissue,and the fracture healed well.The biomechanical experiment showed that the ultimate compressive strength of 12 W femurs was 24.9±0.7MPa.Conclusion: The nano HA/ZrO₂ gradient composite scaffolds prepared by photocoagulation stereoscopic printing technology have good biocompatibility and biomechanical strength,without obvious cytotoxicity.Gradient composite material which was wrapped calcium alginate sustained release system is applied in animal experiments,showing good bone defect repair ability.It is an ideal tissue-engineered bone material.