| OBJECTIVE:Both biological xenogeneic cancellous bone (provided by Guangdong GuanHao biological technology Co., LTD.) and allogeneic bone (deep frozen irradiated rabbit bone) were used for segmental bone defect repairing in rabbits’ radius in this randomized controlled animal experiment to compare their treatment effect and side effect to the host and local tissue, meanwhile, provide an evidence of safety and effectiveness for further clinical trials of biological xenogeneic cancellous bone.METHODS:Thirty-six rabbits whose ages ranged from4to6months and weights ranged from2.5to3.5kg were selected. There is no gender limitations. All rabbits were numbered randomly from No.01to No.36. No.01to No.12were assigned to group A, No.13to No.24were assigned to group B, and No.25to No.36were assigned to group C, then each group contained12rabbits. All the thirty-six animals were experienced an osteotomy in one of their radiuses and became a15mm-long segmental bone defect model. Rabbits in Group A, the experimental group, were implanted with biological xenogeneic cancellous bone, and rabbits in Group B, the experimental control group, were implanted with deep frozen allogenic bone. Group C was the blank control group. For each animal, postoperative observation for general condition was carried and the result was profiled respectively. Four animals were sampled in a systematic random method from each group4weeks,8weeks, and12weeks after operations and experienced anatomy observation, X-ray examination scored by Lane radiographic scoring system, histological examination scored by Lane histology scoring system, and biomechanical experiment. The overall results of radiographic scores were analyze by the method of two-factors completely randomized factorial design (3×3) analysis of variance, Pairwise comparisons between every two groups were carried by the method of Bonferroni test. The overall results of biomechanical experiment were analyze by the method of two-factors completely randomized factorial design (2×3) analysis of variance, and comparisons between groups were carried in the method of Bonferroni test. The overall results of histological scores were analyze by the method of two-factors completely randomized factorial design (2×3) analysis of variance.RESULTS:1. Result of observation under the scanning electron microscopeAppearance:the biological xenogeneic cancellous bone were processed into white or pale yellow sticks in a strip shape of5mmx5mmx40mm without smell. Its trabecular structure was porous and loose, with appropriate toughness and elasticity in wet.View under scanning electron microscope:Biological xenogeneic cancellous bone has a lot of three-dimensional pore, and the natural trabecular structure was preserved. Pore diameters varied from50μm to400μm, and the larger pores were connected by those smaller ones. The porosity percentage was72%～75%. According to the result of observation under electron microscope, this material is an appropriate scaffold with a natural porous and three-dimensional structure, and contains no redundant tissue such as bone marrow tissues or blood cells.2. Result of general observationAll animals awaked in about15min after the operation, and were put back to their cages respectively. Antibiotics were used to prevent infection in the first five days. Their diet, excretion, and activities became normal gradually from the third day to1week, and some protective postures were taken to ease the pain of affected limb. Their incisions were mild swelling in the first3days, then the swelling subsided gradually in4-5days. Most of the incisions have healed in2weeks without obvious secretions and infections, and been covered by new fur in4weeks. All thirty-six rabbits were included in the analysis without missing or death.3. Result of anatomy observationNo necrosis, suppuration, effusion were found in the tissues around the implant of the experimental group or the control group4weeks,8weeks, and12weeks after the operation.4weeks after the operation, xenograft of the experimental group remained intact with its surface covered by host soft tissues. Small amount of callus were found around. The interface between xenograft and host bone began to blur, and the combination between them was reliable to a certain extent; Allograft of the control group also remained complete, but looked pallor compared to host bone nearby. Small amounts of callus were found around and connective tissues coated the implant. The interface between allograft and host bone is clear; The bone defect in blank control group did not repair yet.8weeks after the operation, xenograft of the experimental group remained intact with its pores fulfilled by host soft tissues. Some pannus were found around and the amount of callus increased. The interface between xenograft and host bone became more blur, and the combination between them was more reliable; Allograft of the control group also remained complete and pallor. The amounts of callus increased. The interface between allograft and host bone can be recognized; The bone defect in blank control group did not repair yet.12weeks after the operation, xenograft of the experimental group began to degrade and coated by plenty of pannus with its pores fulfilled by host soft tissues. The amount of callus continued increasing. The interface between xenograft and host bone began to fuse, and the combination between them was firm; Allograft of the control group also began to degrade, but its shape still can be recognized. The amounts of callus increased but no pannus were found around the implant. The interface between allograft and host bone also began to fuse, and the combination between them was reliable; The bone defect in blank control group did not repair yet.4. Result of X-ray examination and Lane radiographic scoring The degree of bone defect repair were evaluated by X-ray examination4weeks,8weeks, and12weeks after the operation, and the Lane radiographic scoring was used to describe the result.On the first of surgery, a low-density implants shadow can be seen in the middle of the radial in the experimental group, the location was perfect, the fracture lines were clearly visible, and the ends of the bone defect were neatly; The shadow in control group had the same density with the host bone, the location was good, the fracture lines were clearly visible, the ends of the bone defects were neatly; A segmental bone defects was observed in the middle of the radial in blank control group. The defect length was15mm, and the ends were neatly.Four weeks after the surgery, the implant density of the experimental group increased compared with the previous images, the shadow of a small amount of callus were visible, edges of the fracture were indistinct, and part of the fracture line were visible; Implants of control group had a similar density with the host bone, cortical bone contours can be seen, a small amount of callus were visible around the fracture lines, the gap widened, the edges of the bone ends fuzzed; The bone defect in blank control group have not yet been repaired, but the edges of the bone ends fuzzed.Eight weeks after the surgery, the implant density of the experimental group continued increasing, the shadow of callus also developed a lot, edges of the fracture were indistinct, and part of the fracture line were hard to recognize; Implants of control group had a similar density with the host bone but a higher density in some points, cortical bone contours still can be seen, the fracture lines and the edges of the bone ends fuzzed; The bone defect in blank control group have not yet been repaired, but the edges of the bone ends seemed more fuzzy.Twelve weeks after the surgery, the implant density of the experimental group continued increasing and some part seemed similar to normal part, a lot of callus developed and connected with host bone, the edges of the fracture were almost disappeared; Implants of control group had a higher density than the host bone, cortical bone contours still can be seen but part of that degraded, the fracture lines and the edges of the bone ends were hard to recognize; The bone defect in blank control group have not been repaired yet.All X-ray images were evaluated according to Lane radiographic scoring system. The overall results were analyze by the method of two-factors completely randomized factorial design (3X3) analysis of variance. The scores increased continuously in4th week,8th week, and12th week and the differences among three groups in were significant, F=85.245, P=0.000. Pairwise comparisons between every two groups were carried by the method of Bonferroni test. The difference between experimental group and blank group and that between control group and blank group were both significant, P<0.05. The difference between experimental group and control group was not significant, P=0.851.The statistical results indicate that both implants of the experimental and control groups can be effective in promoting the healing of segmental bone defect in rabbits’radius, and the biotype heterogeneous cancellous bone was equivalent to deep-frozen allograft bone. The bone defect of blank group have not been repaired.5.Result of biomechanical experiment.Radial specimens of the experimental group and the control group experienced biomechanical experiment4weeks,8weeks, and12weeks after operation. RGM-3010computer controlled biomechanical testing machine was used to conduct the three-point bending experiments in the implanting part of the specimen first, and then, the normal part of the same one. All the results were recorded automatically by computer. The percentage were calculate by the maximum load of implanting part versus that of normal part, and were used to describe the rebuilding degree of biomechanical performance. The test environment temperature is25℃, humidity of50%, a span of20mm, and speed10mm/min. The overall results were analyze by the method of two-factors completely randomized factorial design (2×3) analysis of variance, and comparisons between groups were carried in the method of Bonferroni test. Radial biomechanical strength of both experimental group and control group gradually recovered at4weeks,8weeks,12weeks after the operation but there was no significant differences between the two groups, F=2.492,P=0.132. Statistical results indicate that the biomechanical strength of both experimental group and control group gradually recovered over time, but the bone defects in two groups did not reach the complete reconstruction within12weeks, and the materials were not complete degraded. No significant differences were found between the two groups in the aspect of biomechanical strength recovery.6. Result of histological examination and Lane histology scoringThe degree of bone defect repair of experiment group and control group were also evaluated by histological examination at4weeks,8weeks, and12weeks after the operation, and the Lane histology scoring was used to describe the result.Four weeks after the operation, the cancellous structure of xenograft of the experimental group can still be identified, and tiny blood vessels were found inside the material. Deposition of irregular new host bone and bone-like tissues were also appeared. Osteoblasts, macrophages, and occasionally bone trapfossa structures can also be found, the implant material began to fragmentation, the junction region between materials and autologous bone began to connect. The implant material of the control group remained the original cortical bone structure, but neither osteoblasts survived inside the Lacunae, nor newborn tiny blood vessels. A few of macrophages, inflammatory cells and new bone gathered around the fragmentation of the material.Eight weeks after the operation, the xenograft of the experimental group began to degrade, and more tiny blood vessels were found inside the material,with amount of deposition of irregular new host bone and bone-like tissues. The number of osteoblasts and macrophages increased. The implant material of the control group remained the original dead cortical bone structure without newborn tiny blood vessels. There were more macrophages, inflammatory cells and new bone gathered around the fragmentation of the material. The conjunction area of materials and autologous bone began to reconstruct and transform.Twelve weeks after the operation, the xenograft of the experimental group continued degrading, and a lot of new bone reformed trabecular bone alternated with host bone. Amount of tiny blood vessels were found inside the material. The number of osteoblasts and macrophages increased. The implant material of the control group remained dead cortical bone structure in the center of the allograft but degraded in the junction part. There were more new bone gathered around the fragmentation of the material, but still no newborn tiny blood vessels were found. The conjunction area of materials and autologous bone reconstructed.The results of experiment group and control group were evaluated according to Lane histological scoring system. The overall results were analyze by the method of two-factors completely randomized factorial design (2×3) analysis of variance. The scores increased continuously in4th week,8th week, and12th week, and at the same point of time, the differences between groups were not significant. F=0.194, P=0.665. The statistical results indicate that both implants of the experimental and control groups can be effective in promoting the healing of segmental bone defect in rabbits’ radius, and the biotype heterogeneous cancellous bone was equal to deep-frozen allograft bone.CONCLUSION:1. No obvious immune response or rejection occurred in rabbits implanted with biological xenogeneic cancellous bone or deep frozen allogeneic bone. Of which the heterologous immunity have been eliminated thoroughly, biological xenogeneic cancellous bone are equaled with allogeneic bone in the aspect of biological compatibility.2. Both biological xenogeneic cancellous bone and deep frozen allogeneic bone repaired the segmental bone defect in rabbits’radius eventually.The revascularization process developed faster and earlier in biological xenogeneic cancellous bone than that in deep frozen allogeneic bone. Biological xenogeneic cancellous bone and frozen allogeneic bone tied in the aspect of osteogenic ability and biomechanical performance. |
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