The Preparation Of Antibacterial Modified Eggshell As Scaffolds Material And Experimental Study On Repairing Skull Defects In Rabbit

[Objective]This study is to observe the anti-bacterial complex eggshell powder as scaffold in repairing rabbit’s critical size skull defects and investigate the ability of material in repairing bone defects preliminarily. Explore the feasibility of the anti-bacterial complex eggshell powder as scaffold can be used in situ tissue engineering.[Methods]1、 Avian eggshell (ES) were cleaned and grounded into powder forms,300-500μm in diameter. Surface-modified ES powder and zinc-coated ES powder were prepared by microwave method. The powders were characterized using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDXA), X-ray diffraction (XRD)、Fourier transform infrared spectroscopy (FT-IR).2、Prepared the critical-size cranial defect model:30adult male New Zealand white rabbits were selected for this experiment.5cranial defects (6-mm diameter) were created per rabbit. They were divided into5groups.(group A:the defects were unfilled; group B:the defects were filled with ES powder; group C:the defects were filled with surface-modified ES powder for60min by microwave method; group D:the defects were filled with zinc-coated ES powder; group E: the defects were filled with Bio-OssTM). All the animal models were randomly divided into3groups. The rabbits in each group were sacrificed at postoperative weeks of4,8,12. Before being killed, the rabbits were labelled with fluorescence.3、Bone histomorphometry specimen preparation:Used low-speed saw to cut the skull and saw up the defect models with Scaffolds materials. After fixation, dehydration, infiltration, polymerization, used microtome to make slices along the longitudinal of cranial,8μm slices were used Goldner-Trichrome stain, slices for fluorescent observation were20μm, subtle pathological changes in bone tissue were observed under ordinary and fluorescence microscope, and then using the image analysis equipment for parameter measurement, calculation and analysis.^Data was expressed as mean±standard deviation(SD) and analyzed by SPSS16.0.[Results]1、ES own pore structure as bone tissue. The surface-modified ES with CHA and the zinc-coated antibacterial ES as scaffolds material can be prepared by microwave method.2、The animals recovered well after the surgery. There was no infection and secondary fracture in experimental animals. The gross specimen showed:As time after surgery on, rabbits’bone defect cavities became smaller in experimental groups, without materials out, while bone defect cavities were full of granulation tissues in control group.3、Observation with fluorescence dual-labeling technique of undecalcified bone tissue:In Group A, the fluorescence only can be seen in marginal areas of defects. Many green and yellow fluorescent bands were seen in marginal areas and the implanted sites in group B, C, D and E.4、Goldner-Trichrome stain:relative newly formed bone area and relative residual bone graft particle area were measured and then statistically analyzed. In Group A the amount of new bone formation was a little varied among4,8and12weeks and no statistical differences was found. But in Group B,C,D and E the amount of new bone formation found statistical differences among4,8and12weeks (P<0.05).It showed that newly formed bone increased with the extension of time. The relative newly bone area of defects with different bone graft materials at4,8or12weeks were compared respectively. The statistical differences were found at4,8or12weeks (P<0.05). The new bone area of group A was the least. Compare the residual bone graft particle area of group B, C, D and E between4,8and12weeks. The statistical differences was found (P<0.05).With the time the residual graft particle was biodegradable. There were no statistical differences for group E and it showed the graft particle was not biodegradable obviously. At4weeks the residual bone graft particle area had no statistical differences between group B, C, D and E. But at8and12weeks the statistical differences was found (P<0.05) [Conclusion]ES had excellent biocompatibility and could promote new bone regeneration. The surface-modified ES and the zinc-coated antibacterial ES had a higher biodegradability and osteoconductivity. The surface-modified ES and the zinc-coated antibacterial ES as bone graft material were suitable for in situ bone tissue engineering.