| Objective:Rabbit lumbar posterolateral intertransverse process fusion was established using the composite material A, which compound with the nano-hydroxyapatite(Nano-HA) modified biphasic calcium phosphate ceramics and chitosan(Cs), and composite material B, which loaded recombinant human bone morphogenetic protein-2 (rhBMP-2) on composite A, as spinal fusion bone graft substitutes. The two kinds of composite A and B were implanted to rabbit posterolateral intertransverse process separately. The purpose was to observe the characteristics of bone substitute and expore the mechanism of bone formation of the composite materials. It will provide an animal experimental basis for the further improvement and optimization of the bone graft substitutes.Methods:Forty eight skeletally maturated Japanese white rabbits (age,1～1.5 years-old; weight,3.5～4.5 kg) were randomly divided into three groups depending on the materials implanted to the intertransverse process. The nano-hydroxyapatite/biphasic calcium phosphate ceramics/chitosan composite was implanted to group A, and the nano-hydroxyapatite/biphasic calcium phosphate ceramics/chitosan/recombinant human bone morphogenetic protein-2 to group B and autogenous iliac bone graft to group C. For each group there were sixteen rabbites. Each rabbit underwent surgery for a single level bilateral posterolateral intertransverse process fusion at L5-L6.X-ray and CT scan was done to obersve the bone healing separately at 4 and 8 weeks after surgery. After radiographic examination at 8 weeks postoperation, all the experimental animals were killed to observe the posterolateral fusion mass by general observation, manual palpation and histologic examination.Results:1. Experimental animals were generally in good condition after surgery. The day after operation, all animales sobered and started drinking water, started eating the next day. The wound was I/A healing without evidence of bleeding, swelling, skin effusion and purulent infection complications. There were also no neurological complications, implant rejection, and allergic reactions.2. According to the radiographic evaluation criteria established by Curylo, four weeks after operation X-ray showed that the overall solid fusion rate of the three groups was statistically significant difference (P＜0.05). The fusion rate of group C was higher than that of group A and group B, but the difference between group A and group B was not statistically significance (P＞0.05). Eight weeks postoperation the overall solid fusion rate between the three groups was statistically significant difference (P＜0.05).The fusion rate of group C was higher than that of group A and group B; There was significant difference between group A and B(P＜0.05). The fusion rate of group B was higher than that of group A.3. Four weeks postoperation CT scan revealed that there was no fusion in group A and only one case was assessed as solid fusion in group B and twelve in group C. The overall solid fusion rate between the three groups was statistically significant difference (P＜0.05). But there was no statistically significant difference between group A and group B (P＞0.05). The difference between group B and group C was statistically significance (P＜0.05), and so it is between group A and group C(P＜0.05). Eight weeks after operation CT scan showed that there was no fusion case in group A and much of the group B did not get solid fusion (11 cases). The solid fusion rate of group B was much lower. Most specimens of the group C got solid fusion (15 cases). The overall comparison of the three group was statistically significant difference(P＜0.05). There was statistically significant difference between group A and group B (P＜0.05); Group B and group C were statistical difference (P＜0.05); There was statistically significant difference between group A and group C (P＜0.05). There were a large number of high density materials remained within the composite of group A and B. 4. Eight weeks after operation gross observation showed that the composite material of group A combined with the surrounding muscle tissue closely. No fibrous capsule wraping the materials and cavity formation was not found. There was no infection, necrosis, purulent, fluid phenomena around the implant materials. Materials still remained in the intertransverse process. The materials degraded partly and the pore size on the surface increased.There was much tissue that grew into the holes. There was also no infection, necrosis, purulent, fluid phenomena around the implant materials of group B. The materials closely integrated with the surrounding muscle tissue, without fibrous capsule wraping the materials and cavity formation. Four cases showed obvious sign of fusion. Materials still remined between the transverse process in the rest specimens, with plenty of tissue grew into the holes too. The grafted autogenous iliac bone combined with the surrounding tissue closely. Boney connection can be seen between transverse process and the grafted bone, with obvious callus formation. The fusion mass between the transverse process was hard.5. Eight weeks postoperation manual palpation showed that no one sample got fusion in group A. Only four specimens of group B got solid fusion, with much lower rate of fusion. The majority of group C(15 cases) got solid fusion. The overall solid fusion rate between the three groups was statistically significant difference (P＜0.05). There was significant difference between group A and B (P＜0.05). Group B and group C were statistical difference (P＜0.05). Also, There was statistically significant difference between group A and group C (P＜0.05).6. Eight weeks after the surgery histologic examination showed that there was part of degradation in the composite of group A. A large number of capillaries formed onto the surface and grew into the inner of the materials of group A. No fiber capsule formed between materials and surface tissue, and a small amount of osteoid formed within the material, which performed good biocompatibility and a certain bone conductivity. There was also part of degradation in the composite of group B. No inflammatory reaction was seen on the surface and inner of group B which showed better compatibility with the surrounding tissue. The junction of the materials and transverse process was connected by bony component. Newly formed woven bone was found inside the composite of group B, but mature bone and bone marrow did not appear. The composite materials of group B have a certain degree of bone induction ability. A large number of newly formed bone was seen in group C. A lot of matured bone and bone marrow appeared. The autogenous bone has much better bone conduction, bone induction and bone formation ability. Conlusions:1. Use nano-hydroxyapatite modified biphasic calcium phosphate ceramics compound chitosan as the carrier of rhBMP-2 for lumbar posterolateral intertransverse process fusion in rabbits. Load a much lower dose of rhBMP-2 (30μg/side) to induce a certain degree of new bone formation at the inner of the composite.2. The Nano-HA/BCP/Cs/rhBMP-2 composite has an excellent biocompatibility, a certain degree of bone conductivity and bone induction that can induce new bone formation within the materials after implantation. Bone formation is better than that of Nano-HA/BCP/Cs group. But compared with the autologous bone, there are still some disadvantages. To be good bone graft substitutes, Nano-HA/BCP/Cs/rhBMP-2 composite requires further improvement.3. The Nano-HA/BCP/Cs composite can be degraded and absorbed in vivo when implanted into the body. The composite itself and its degradation products do not have adverse effects on test animals. There were plenty of capillaries that grew into the material and a small amount of osteoid formed inside the materials. Therefore the composit materials have much better biocompatibility and some degree of bone conductivity.4. Eight weeks after implantation of the two groups of composite materials, a majority of residual materials were observed in the fusion mass. New bone formation and material degradation rate does not match better enough. Because of the remained high-density HA, it is much difficult to assess weather it is fusion or not in the fussion mass. The material need to be further optimized.
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