新型梯度复合HA/ZrO2组织工程骨支架在猕猴颈椎融合中的应用  

Application of a novel gradient HA/ZrO2 engineering bone scaffold for repair of cervical fusion in rhesus macaque

作　　者：邵荣学[1,2] 全仁夫[1] 王拓[1] 杜伟斌[1] 王栋[2] 贾高永 吕龙宝[3] 韦习成[4] 杨贺杰[2] 陈惠国[2] 乐军[2] 潘浩[2] 周辉[2] 杨迪生[5] 

机构地区：[1]浙江中医药大学附属江南医院脊柱外科,杭州311201 [2]杭州市中医院骨伤科,杭州311201 [3]中国科学院昆明动物研究所 [4]上海大学材料与工程学院 [5]浙江大学医学院附属第二医院

出　　处：《中华解剖与临床杂志》2017年第6期499-509,共11页Chinese Journal of Anatomy and Clinics

基　　金：浙江省重大科技专项（2014C03031）;浙江省科学技术厅公益技术研究社会发展项目（2012C33114）;杭州市重大科技专项（20122513A14）.

摘　　要：目的 构建新型梯度复合羟基磷灰石-二氧化锆（HA/ZrO2）组织工程骨支架,并评估其在猕猴颈椎椎间融合中的效果.方法 通过离子交联法制备壳聚糖水凝胶作为骨形态发生蛋白2（BMP-2）的缓释载体,扫描电镜下观察其微观形态,检测其载药量、包封率及缓释速率;然后分离、培养猕猴源性骨髓间充质干细胞（BMSCs）,并进行碱性磷酸酶、冯库萨染色等对其进行鉴定;最后将前期制备好的BMP-2明胶/壳聚糖凝水胶缓释系统和第3代猕猴BMSCs加载于HA/ZrO2泡沫陶瓷,以构建新型组织工程骨,并观察其形态特征.将24只雄性猕猴按照随机数字表法分成4组,其中组织工程骨组（n=8）为植入梯度复合HA/ZrO2泡沫陶瓷负载BMP-2明胶/壳聚糖水凝胶缓释系统和第3代猕猴BMSCs,泡沫陶瓷组（n=8）为植入梯度复合HA/ZrO2泡沫陶瓷,自体髂骨组（n=4）为植入自体髂骨,假手术组（n=4）为只对相应部位的软组织进行切开、缝合,未破坏其椎间盘;观察术后颈椎X线（术后即刻、8周、16周）、组织形态学表现（术后8周、16周）及测试相应节段的生物力学（术后16周）.结果 扫描电镜下BMP-2明胶/壳聚糖水凝胶呈3D网状结构,内见均匀分布的壳聚糖微球,其负载BMP-2后的包封率和载药率随时间逐渐降低：第1天分别为87.4%＆#177;0.9%和58.2%＆#177;0.5%、第15天分别为45.2%＆#177;0.6%和30.1%＆#177;0.4%,累积释放率第1天为12.6%＆#177;0.11%、第15天为55%＆#177;0.16%.显微镜下观察见BMSCs的形态呈多样性,以梭形及短棒形等较常见;第3代猕猴BMSCs成骨诱导后的碱性磷酸酶、冯库萨染色以及表面特异性抗原的检测结果显示,符合BMSCs的生物学特性.不同时点的X线及组织形态学观察显示材料内部新生骨量组织工程骨组较泡沫陶瓷组明显增多;生物力学测试结果显示,在最大载荷、抗压强度和能量吸收方面假手术组均低于其他3组（P值均〈0.05�Objective To prepare the gradient HA /ZrO2 engineering bone scaffold and evaluate its bone repair capability in the cervical fusion of rhesus macaque .Methods Bone morphogenetic protein 2 （BMP-2） in gelatin/chitosan slow-release hydrogel was prepared by multiple emulsion cross -linking, and its biological features were examined .Mesenchymal stem cells （ BMSCs） of rhesus macaque were isolated ,cultured and identified.The novel gradient HA /ZrO2 engineering bone scaffold was prepared for carrying BMP-2 and BMSCs, and its morphological characteristics were observed .The cervical intervertebral disc of experimental segments in 24 healthy male rhesus macaques were replaced with the novel gradient HA /ZrO2 engineering bone scaffold carrying a gelatin /chitosan gel slow-release hydrogel loading BMP -2 and 3rd generation of BMSCs （group A, n =8）, or the empty novel gradient HA /ZrO2 engineering bone scaffold （group B, n =8）, or autologous iliac graft （ group C, n =4） or nothing （ group D, n =4）.The anteroposterior and lateral radiographs of the cervical spine were taken at once , postoperative 8 and 16 weeks.Histomorphometry was performed on samples at postoperative 8 and 16 weeks, and biomechanical testing was performed on harvested vertebral samples 16 weeks after operation.Results Electron microscopy for gelatin/chitosan gel slow-release hydrogel loading BMP-2 revealed a three-dimensional network structure with uniformly distributed chitosan micro -spheres, and its encapsulation efficiency and drug loading rate of BMP-2 were decreased with time, 87.4% ＆#177;0.9% and 58.2% ＆#177;0.5% on day 1st respectively, 45.2% ＆#177; 0.6% and 30.1% ＆#177;0.4% on day 15th respectively.The release rates of BMP-2 were 12.6% ＆#177;0.11% and 55% ＆#177;0.16% on day 1st and 15th respectively.BMSCs with diverse cell morphology were found under microscope.After osteogenic induction of the 3rd generation BMSCs, the results of alkaline phosphatase , von Kouza staining and its surface-specific antigen tests

关 键 词：组织工程 颈椎 生物相容性材料 猕猴 间质干细胞 成骨细胞 骨形态发生蛋白质2 骨再生 羟基磷灰石-二氧化锆 动物实验 Bone morphogenetic protein 2 

分 类 号：R687[医药卫生—骨科学]