| Every year, bone defect for a variety of reasons, such as injury, infection, diseases, and many others, desires for bone repair. In the United States, there are about 500,000 surgical bone repairs, and over 300,000 spinal fusions are conducted one year. Although autologous bone is considered the gold standard for bone repair, but the complications associated with the donor site reach 30% and limitation amout of bone limited its applications. Synthetic materials are considered the most promising bone graft substitute materials. HA and BCP due to good biocompatibility and bioactivity have been widely used in clinical and scientific study for bone repair and spinal fusion. Compared with HA, BCP has higher biological activity, biocompatibility and biodegradability, and promoting new bone formation and ingrowth, promoting bone integration with host bone. Compared with ordinary HA, the more excellent bioactivity in nanoHA is conducive to cell adhesion, osteogenic differentiation, bone ingrowth and integration. In vitro and in vivo studies have shown electromagnetic fields can accelerate osteogenic differentiation, proliferation, and new bone formation. Electromagnetic fields stimulation combined with artificial bone, can improve the bioactivity and biocompatibility of artificial bone. Combined magnetic field has been applied in clinical treatment of fractures, nonunion and delayed union for over 10 years, and in promoting spinal fusion too.Since the bioactivity and biodegradability of BCP is higher than HA, and nanoHA has ability of significantly promoting osteoblast differentiation, cell adhesion, proliferation, and calcium deposition, therefore, we hypothesize that the bioactivity and biocompatibility in nanoHA coating porous BCP artifical bone (AB) will be higher than the pure porous BCP. Combination CMF stimulation with the novel nanoHA /BCP artifical bone for PLF in rabbit model can further improve the spinal fusion rate. The study included the following two parts.Part1 Preparation of Novel nanoHA Coating Porous BCP Artificial BoneOBJECTIVE To preparate a novel nanoHA coating prous BCP artificial bone by several coating methods. To compared advantages and disadvantages of the coating methods.METHODS The nanoHA coatings of porous BCP artificial bone were prepared by hydrothermal chemical deposition method, biomimetic method and the biomimetic method under ultrasonic radiation (40KHz). The porosity, water absorption and density of the AB were measured by means of drainage.The mechanical properties of the AB were measured by three-point bending test and compression test. The morphology, crystallinity and phases of the coating were studied in detail by means of FESEM, TEM and XRD. RESULTS (1) Compared with precoating by hydrothermal chemical deposition, the porosity, water absorption and density in porous BCP artificial bone were significantly different. HA crystals layer was prepared on the outer and inner surface of porous BCP artificial bone by hydrothermal chemical deposition method. The coating of the glass-phase porous BCP artificial bone was constituted of the short rod-like nanoHA crystals with an average diameter of 58.3nm,100-400nm in length. The coating of the crystal-phase porous BCP artificial bone was constituted of micron hexagonal HA crystals, the average diameter was 434.8nm, and the length was between the 1000nm and 3000nm. The Young’s elastic modulus, maximum bending load and compressive strength of the glass-phase porous BCP artificial bone were mildly improved by the treatment of hydrothermal chemical deposition(P>0.05). On the contrary, the mechanical properties of the crystal-phase porous BCP artificial bone were significantly improved (P<0.05). However, the mechanical properties of glass-phase porous BCP artificial bone were significantly superior to the crystal-phase porous BCP artificial bone (P<0.05).(2) Porous BCP artificial bone soaked in SBF formed flower clusters like micron HA crystal on itself, the average diameter of HA crystal was 142.2nm, the length of 2μm or more, the amount of HA formation was in time-dependent pattern. There were not significant effects on the HA formation on porous BCP artificial bone under 40KHz ultrasonic radiation (P>0.05).CONCLUSIONS (1)NanoHA coating glass-phase BCP atificial bone was successfully prepared by hydrothermal chemical deposition method. The mechanical strength was mildly improved.(2) Micron-HA coating crystal-phase porous BCP artificial bone was successfully prepared by hydrothermal chemical deposition method. The mechanical strength was significantly improved.(3) The amount of micron HA crystal formation on the porous BCP artificial bone in SBF was less and in time-dependent pattern,40KHz ultrasonic radiation can not accelerate HA formation porous BCP artificial bone in SBF. (4) The nanoHA coating porous glass-phase BCP artificial bone developed in this study with better biomechanical properties can be serve as a promising alternative to bone graft material for bone tissue engineering.Part 2 Effects of CMF on nanoHA/BCP for PLF in Rabbits: an Experimental StudyOBJECTIVE To investigate effects of nanoHA Coating and CMF treatment on rabbits PLF, observe the effects of CMF treatment combined with nanoHA/BCP for PLF in rabbits, and explore the mechanism and principles of CMF and nanoHA coating in spinal fusion so as to provide a scientific basis for the future clinical applications. METHODS 48 adult rabbits undergone bilateral intertransverse process fusion at the level of L5-6 without internal fixation were randomly divided into six groups G1, G2, G3, G4, G5 and G6 according to grafts used and CMF treatment. group G1 received autologous iliac bone graft(AIBG) and CMF treatment; Group G2, nanoHA/BCP+CMF treatment; Group G3, BCP+CMF treatment; G4 groups:AIBG+placebo; G5 Group:nanoHA/BCP +placebo; G6 Group:BCP+placebo. CMF treatments were performed at 1 week after surgery for 8 weeks,30 minutes a day. The animals were euthanized at 9 weeks after surgery, and the following post-mortem analyses were performed. (1)Evaluating spinal fusion rate by palpation; (2)Radiographical evaluation including spinal fusion and fusion mass normalized optical density index;(3) High-resolution CT scanning evaluation including the spinal fusion and grafts morphology; (4)FESEM evaluation including the gap between AB and host bone, the morphology of AB, and new bone formation and collagen mineralization in pore; (5)Three-point bending test evaluation including the bending stiffness, maximum stress and maximum bending load; (6)Histological analysis with decalcified sections stained with H&E and undecalcified sections stained with toluidine blue including spinal fusion, new bone ingrowth radio(BGR) and artificial bone residual radio(ABRR). BMP-2 and TGF-β1 expression in fusion mass were studied by immunohistochemistry; (7) The crystallinity and phases of AB at 9 weeks after surgery were studied by XRD.RESULTS (1)The evaluation of spinal fusion rate by palpation, radiography and histology showed the highest fusion rate in group G2, the lowest in G6, there was statistical difference between the two groups(P<0.05). Factorial analysis showed that CMF and nanoHA coating significantly improved the spinal fusion rate (P<0.05). (2) The normalized optical density index of fusion mass in group G2 was significantly higher than the other groups (P<0.05), factorial analysis showed CMF and the nanoHA coating significantly increased the normalized optical density index (P<0.05).(3) CT, FESEM, histological observation showed that the AB were covered with new bone callus and bone ingrowth. Continuous bone bridge formed between the L5 and L6 transverse process in some specimens. (4)FESEM showed the gap between AB and host bone was more smaller in group G2 than other groups, and signicantly smaller than group G6(P<0.05). Mineralization rates of collagen in AB in group G2 were the highest in all groups, statistically significantly higher than the group G6 (P<0.05). nanoHA coating significantly increased the collagen mineralization in the AB pore.(5) Histological resruts showed, compared with others there was more new bone ingrowth achieved throughout the implanted nanoHA/BCP via interconnecing pores and excellent unification between the AB and the newly mineraliaed bone in group G2. Compared with group G2, the BGR in group G3, G5 and G6 was significantly lower (P< 0.05), the BGR in group G6 was significantly lower than the group G4 and G5 (P=0.039, P=0.052). Factorial analysis showed that CMF treatment increased the BGR by 10.31% (P<0.05), nanoHA coating significantly increased BGR by 9.87%(P<0.05). Histological fusion score of group G2 was significantly higher than that of group G3, G4 and G6 (P<0.05), group G1 in histological fusion score was significantly higher than that of group G6 too (P<0.05), there was not statistically difference among the remaining groups. Factorial analysis showed that CMF and nanoHA coating significantly improved fusion scores. Immunohistochemistry showed that CMF significantly increased the BMP-2 and TGF-β1 expression level in fusion mass (P<0.05), but nanoHA coating did not influence their expression(P> 0.05). (6) Biomechanical test showed the bending stiffness in group G2 was significantly higher than the other groups (P<0.05). Bending stiffness in group G5 was also significantly higher than the group G4 and G6 (P <0.05), G1 Group Significantly higher than the group G4 (P<0.05). Factorial analysis showed that CMF significantly improved the bending stiffness by 20.41 N/mm (P< 0.05), the grafts factor significantly increased the bending stiffness by 23.85 N/mm (P<0.05). (7) XRD phase analysis showed that crystallinity decreased after AB implanting. The proportion of HA of nanoHA/BCP scaffolfd declined, but BCP artificial bone increased in the proportion of HA.CONCLUSION (1) The novel nanoHA coating porous BCP artificial bone developed in this study with good biocompatibility and bioactivity and biodegradability can be serve as a promising alternative to bone graft material for spinal fusion. (2) Both CMF and nanoHA coating can promote new bone formation and osseointegration, improve the spinal fusion rate and the biomechanical properties of the fusion mass in this rabbit PLF model. CMF can improve the biocompatibility and bioactivity of artificial bone. (3) CMF combined with nanoHA/BCP for PLF in rabbits can significantly improve the fusion rate, better than the pure autologous bone fusion, which can be used as a new spinal fusion method. |
