摘要
目的 探讨BMP-2明胶/壳聚糖水凝胶缓释系统复合骨髓间质干细胞种植于HA/ZrO2泡沫陶瓷构建的组织工程骨修复猕猴腰椎椎体缺损的疗效.方法 将BMP-2明胶/壳聚糖水凝胶缓释系统和第3代猕猴骨髓间质干细胞加载于HA/ZrO2泡沫陶瓷,构建组织工程骨,观察其形态特征.制作猕猴L4或L5椎体缺损模型24个,分别植入组织工程骨（n=10）、泡沫陶瓷（n=10）、自体髂骨（n=4）.术后8周、16周观察腰椎脊柱X线、Micro-CT扫描及组织形态学表现,术后16周行相应节段的生物力学测试.通过RT-PCR和Western Blot检测组织工程骨组和泡沫陶瓷组的Ⅰ型胶原、骨钙素、骨桥蛋白、碱性磷酸酶、碱性成纤维细胞生长因子及血管内皮生长因子的表达.结果 扫描电镜下BMP-2明胶/壳聚糖水凝胶均匀分布于材料及孔隙处,在HA/ZrO2泡沫材料的孔内可见被水凝胶包裹的骨髓间质干细胞;HE染色可见材料表面均匀分布散在的骨髓间质干细胞;BrdU染色观察到黏附于材料表面的细胞生长正常.不同时点的X线、Micro-CT扫描及组织形态学观察显示组织工程骨组材料内部新生骨量较泡沫陶瓷组明显增多;生物力学测试结果显示泡沫陶瓷组的最大载荷和弹性模量低于其他各组,组织工程骨组与自体髂骨组的极限抗压强度和最大吸收能量均高于泡沫陶瓷组（P＜ 0.05）.组织工程骨组Ⅰ型胶原（1.682±0.031）、骨钙素（1.04±0.032）、骨桥蛋白（1.01±0.026）、碱性磷酸酶（1.24±0.023）、碱性成纤维细胞生长因子（3.67±0.018）及血管内皮生长因子（2.36±0.003）基因相对表达量均高于泡沫陶瓷组（分别为1.01±0.012、0.56±0.018、0.42±0.012、0.98±0.036、1.04±0.048、0.62±0.024,P＜0.05）.Western Blot检测结果与PCR一致.结论 BMP-2明胶/壳聚糖水凝胶缓释系统复合第3代骨髓间质干细胞种植于HA/ZrO2泡沫陶瓷构建的组织工程骨能有效修复猕猴椎体缺损,在影像学及组织学表现、成骨相关蛋白检测和生物力学测试方面可达到与自体骨相似的骨替代作用.
Objective To examine the bone regeneration capability of a novel bone graft substitute （BGS） of porous gradient HA/ZrO2 carrying BMP-2 and BMSCs in gelatin/chitosan slow-release hydrogel （BBGC-h） to lumbar vertebral body defect in rhesus monkey.Methods The BBGC-h was evaluated by scanning electron microscope for its morphological characteristics.The BGS with or without BBGC-h was implanted into the lumbar vertebral body defects in rhesus monkeys.Twenty-four healthy rhesus monkeys （5-7 years,5-8 kg） were provided by the Kunming Institute of Zoology,Chinese Academy of Sciences.They were randomly divided into groups A （Test,BGC_BBCG-h,n=10）,B （Control,BGC,n=10） and C （Autologous,n=4）,respectively.The anteroposterior and lateral radiographs of the lumbar spine were conducted under anesthesia at 24 h,8 weeks,and 16 weeks postoperatively.Micro-CT and histomorphometry were performed on samples collected from 2 of 10 samples in each group A and B to observe the newly formed bone volume at 8 and 16 weeks postoperatively.At 16 weeks postoperatively,bone matrix composition markers,including type Ⅰ collagen,osteocalcin,osteopontin,basic fibroblast growth factor,alkaline phosphatase and vascular endothelial growth factor,were analyzed for groups A and B by RT-PCR and Western Blot.Biomechanical testing was performed on harvested vertebral samples of 4 animals from each of the subgroups and 4 additional non-operated healthy animals.Results The BGS revealed a three-dimensional network structure with uniformly distributed chitosan micro-spheres on the surfaces of the material and the interior of the pores.Hydrogel encapsulated BMSCs were visible in the pores of the BGS.The newly formed bone volume in the interior of the pores in the group A was significantly higher than that in the group B at the two time points after surgery via Micro-CT scanning and histomorphology.The results of biomechanical testing indicated that the vertebral body compression strength was the smallest in the group B compared with those of the other groups and the non-operated healthy group （P 〈 0.05）,while the energy to maximal load of group A and group C were higher than those of group B.There were significant higher protein expression of type Ⅰ collagen,OCN,OPN,bFGF,ALP and VEGF in group A than in group B （P 〈 0.05）.Conclusion There may be a promising to use BGS_BBGC-h to repair vertebral bone defects.The novel bone graft substitute has similar effects compared with autologous bone.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2016年第19期1243-1253,共11页
Chinese Journal of Orthopaedics
