Application of novel HA/ZrO2 gradient composite materials made by three-dimensional print technology for repair of femoral shaft defect in dogs

Abstract

Objective To apply the hydroxyapatite/zirconium oxide(HA/ZrO2) gradient composite material by 3D printing technology, to analyze its ability to repair the defect of femoral shaft of beagle dogs. Methods Bone defect model was prepared in 6-month male beagle by truncating right leg femur after middle full-thickness 15 mm, which was put into the Micro CT scanned for volume, complete data collection, transformation and the post-processing. Then data were imported in CeraFab 7500 photocure 3D printer. Started to print program, formed a composite photosensitive resin in the early embryo, and the further defatted sintering and then used dip-coating HA/ZrO2 gradient composite materials according the parameters. The performance of the HA/ZrO2 gradient materials were taken scanning electron microscopy (SEM), X-ray diffraction analysis and biomechanical experiments. Prepared HA/ZrO2 gradient composite materials and cultured L929 mouse fibroblast cell lines, then MTT method was used to detect the HA/ZrO2 gradient composites cell toxicity in vitro. Sixteen dogs were divided into 4 groups, 4 dogs in each group. Group A: Dogs’ 15 mm middle femur were intercepted with no biological material implanted, as the blank control group; group B, C, D were truncated middle femoral 15, 25 and 35 mm made of bone defect model, and transferred into the corresponding specifications of the HA/ZrO2 gradient composite materials. X-ray scan was taken to observe implanted biomaterials combined with own bone and surrounding callus growth after operation of 2, 4, 8, 12 weeks. Animals were executed and captured the entire length of the femur, then observed specimens implanted in biological materials and the surrounding bone growth conditions after 12 weeks. New bone mass were measured and reconstructed by Micro CT scans. The compressive experiments of femoral specimens were used to measure the ultimate compressive strength. Results The SEM showed that nano HA/ZrO2 gradient composite material made by 3D printing technology had smooth surface and even stable structure. Composite material had a metallurgical bonding and there was no obvious boundary in fracture surface. XRD analysis showed an obvious peak, better crystalline degree and better purity. Mechanical text showed that the ultimate compressive strength was (43.37±2.31) MPa. MTT analysis proved that HA/ZrO2 gradient composite material had no cytotoxicity. Front and lateral X-ray examinations were taken at 2, 4, 8, 12 weeks after operation. In group A, bone nonunion was formed. In group B, continual bony callus were get through and there was no obvious boundary between artificial prosthesis and host bone. At 2, 4 and 8 weeks after operation, continuous callus and new bone growth in group C was slower than group B, but the gap between artificial prosthesis and broken ends was gradually filled with new bone, and the continual bony callus were got through at the week 12. In group D, new bone was formed in a lower speed, and appeared only around the broken ends. The specimens were taken at week 12 after operation, three-dimensional reconstruction of Micro CT showed that per unit volume new bone in group B, C, D were (238.55±19.11) mm3/cm3, (223.31±13.41) mm3/cm3 and (110.83±6.48) mm3/cm3, respectively. There were statistically significant differences among the three groups in statistics analysis (F=156.824, P 0.05). Conclusions The nano HA/ZrO2 gradient composite material artificial prosthesis made by 3D printing technology has reliable biocompatibility and biomechanics that fit the individual treatment principle in clinic. As an ideal substitute for bone tissue, it could well repair femoral bone defect in 35 mm in dogs. Key words: Tissue engineering; Hydroxyapatites; Zirconium; Three dimensional printing technology; gradient composite materials; Animal experimentation; Bone defects