Experimental Study Of Individualized Treatment Of Large Segment Bone Defect By Using Nano HA/ZrO₂ Gradient Compsite Materials That Made By Three Dimensional Print Technology

Objective:1.Preparation of calcium alginate microspheres as the release carrier which encapsulating vascular endothelial growth factor 165(VEGF165),and verification of carrier’s release efficiency.2.Prepareation of nano Zirconia-Hydroxyapatite(HA/ZrO2)gradient composite materials by 3D printing technology,and build the co-culture system that composed of calcium alginate release system and HA/ZrO2 gradient composite materials.Implanted this material into dog femoral defect models and study its bone repairing ability.Method:1.Prepared calcium alginate release carrier by cross linking method,respectively crosslinked 12.5 mg/ml,25.0 mg/ml,50 mg/ml concentration of alginate(Alg)with 11.3 mg/ml,22.6 mg/ml,45.2 mg/ml concentration calcium chloride(CaCl2),then lyophilized by freeze-drying apparatus.Scanning electron microscopy was used to observe lyophilized calcium alginate’s microscopic surface morphology of each group,and test swelling coefficient in each group.Used the calcium alginate with minimum swelling degree to be loaded with VEGF165.Enzyme-linked immunosorbent assay kit(ELISA)was employed to detect drug loading mass,encapsulation efficiency and sustained release characteristics of the lowest swelling degree group of calcium alginate which loaded with VEGF165.2.Use Micro CT scanning technology combined 3D printing technology to build Beagle dog femoral shaft defect area nano HA/ZrO2 gradient composite prosthesis,analyzed composite materials’ property by X-ray diffraction(XRD)scanning election microscopy(SEM)and mechanical text.Built co-culture system of calcium alginate encapsulated VEGF165 and composite materials,implanted into defect areas.Divide 16 dogs into 4 groups according to the interception range of femoral shaft,4 dogs in each group.Group A:After the interception of 25 mm middle femur of dog,with no biological material implanted,as the blank control group;Group B:After the interception of 25 mm,with HA/ZrO2 gradient composite materials which composed calcium alginate encapsulating VEGF165 implanted,as the 25 mm femur shaft defect experimental group;Group C:As the experimental group of 30 mm middle femur of the dog was intercepted in the same way;Group D:As the experimental group of 35 mm middle femur of the dog was intercepted in the same way.After operation,morphologically observed the changes of strength,gait and wound of each group;Take X-ray imaging observations respectively at week 6 and week 12 of each group;Animals were sacrificed and taken specimens at week 12 after operation.In vitro Micro CT scan was used to detect the amount of new bone volume inside the material,then the mechanical property was evaluated by femur shaft extreme compression test.Finally,provide a comprehensive evaluation about repairing capability of the novel HA/ZrO2 gradient composite VEGF 165 biological composite materials.Results:1.The hydrogel cross-linked by 12.5mg/ml concentration of Alg and 45.2mg/ml concentration of CaCl2 exhibited the lowest swelling degree,the swelling degree was(207.21%±26.81%),when compared with other groups,there were significantly statistical differences(P<0.05).When loaded with VEGF 165,chitosan hydrogel encapsulation efficiency was 62.38%±3.57%,n=3;The loaded mass of drug was 23.55 ng/mg±2.94 ng/mg,n=3;The release rate was 22.02%±1.36%in 1 d,40.48%±2.56%in 4 d,82.49%±2.51%in 10 d and 85.05%±1.71%.2.The structure of nano HA/ZrO2 gradient composite material made by 3D printing technology was stable,and the distribution was gap.XRD analysis showed an obvious peak,better crystalline degree,better purity.SEM showed that the composite materials had better accuracy,better structure than calcined HA/ZrO2 gradient composite materials.Mechanical text showed the compression strength were 43.37±2.31 MPa.MTT analysis proved the materials had no cytotoxicity.The morphologic observation after operation showed that all the animals wound were healed,no infection and purulent exudation.X-ray examination showed showed at week 4,week 8,week 12 after operation:In group A,with the passage of time after vertebral replacement surgery,callus formation gradually increased around the material.At the week 8,the broken ends were absorbed,callus hypertrophy and bone nonunion formed.In group B,since the gap between artificial prosthesis and broken ends was gradually filled with new bone,so boundary become increasingly blurred.While the artificial prosthesis and autologous bone had almost closely linked at week 12 with continual bony callus getting through and no obvious boundary between artificial prosthesis and host bone;In group C:The early phase was slow than compared with the A group,rupture of the prosthesis appeared at the week 4 after operation,the gap between artificial prosthesis and broken ends was gradually filled with new bone,and there was continual bony callus getting through at the week 12.In group D:New bone formed in a lower speed,and appeared only around the broken ends.The boundary became increasingly blurred.No new bone was found in the middle part of prosthesis at the week 12 after operation,only a small amount of callus was formed around the broken ends and prosthesis.The specimens were taken at week 12 after operation,three-dimensional reconstruction of Micro CT showed that there could be seen a large number of new bone formation inside the prosthesis of group B and group C.The bone volume detection result showed the new bone mass in group B was 238.55±15.61 mm3.In the group C,the new bone mass was 223.31±10.97 mm3,and in group D was 110.48±8.92 mm3.There was no significant difference between group B and C in statistics analysis(P>0.05).The group B and group C were significantly different from group A in statistics analysis(P<0.05).Ultimate compressive test in week 12 showed the compressive strengths of femur shaft were 49.72±2.33 MPa in group B,49.81±2.43 MPa in group C,46.92±3.61 MPa in group D.There was no significant difference between group B,C and D in statistics analysis(P>0.05).Conclusion:The nano HA/ZrO2 gradient composite material artificial prosthesis made by 3D printing technology have reliable biocompatibility and biomechanics that fit individualized treatment principle in clinic.And while composed with the calcium alginate loaded with VEGF165 release system,it can well repair the canine femoral bone defect as an ideal substitute for bone tissue.Its critical value is between 25 and 35 mm.