携载人重组骨形态形成蛋白2及脂肪基质细胞的仿生骨膜构建及强化兔桡骨的再生能力

Constructing mimetic periosteum with recombinant human bone morphogenetic protein 2 and adipose derived stromal cells for the regeneration of radius in rabbits

目的:从仿生学角度出发,首次将脂肪基质细胞、人重组骨形态形成蛋白2、壳聚糖/胶原蛋白/β磷酸三钙膜材基质有机的结合,体外构建仿生骨膜,探讨仿生骨膜修复兔桡骨缺损的可行性,分析其生物相容性及骨再生特点。方法:实验于2005-05/2006-11分别在新加坡国立大学组织工程研究中心、天津医科大学完成。材料制备:成骨诱导化培养兔脂肪①基质细胞、制备释放人重组骨形态形成蛋白2的壳聚糖/胶原蛋白/β磷酸三钙膜材基质,联合生物凝胶+动态种植,三维方式构建仿生骨膜。②实验动物:72只新西兰兔随机分为3组:移植仿生骨膜组,移植诱导骨膜组,空白支架组。③实验方法:建立兔右侧桡骨10mm骨缺损模型,将制备的携载人重组骨形态形成蛋白2及脂肪基质细胞的仿生骨膜、携载人重组骨形态形成蛋白2的诱导骨膜、壳聚糖/胶原蛋白/β磷酸三钙膜材支架分别移植到各组骨缺损区域。④实验评估:术后第2,4,6,8,10,12周观察桡骨移植区域,了解新骨生长及重建情况。苏木精-伊红染色及Masson染色,观察骨组织修复情况。免疫组织化学观察骨形态形成蛋白的表达。双能X射线吸收法测量骨矿物含量、骨密度值。术后第12周,生物力学测试检查骨结构强度力学指标和刚度指标。结果:72只新西兰兔均进入结果分析。①大体观察显示:仿生骨膜组第6周有大量新生骨痂,第12周呈现骨性连接;诱导骨膜组骨质仅部分连接;空白支架组部分骨痂呈向心性生长。②组织学观察情况:仿生骨膜组第6周存在膜内成骨为主的大量新骨,第12周,恢复正常骨结构;诱导骨膜组存在软骨内成骨为主的新骨,第12周,缺损基本修复;空白支架组部分板层骨形成;各组均未见炎性及免疫排斥反应。③免疫组织化学观察:仿生骨膜组增生骨内膜、骨痂骨细胞、骨髓腔骨形态发生蛋白表达显著;诱导骨膜组软骨基质及部分骨膜出现骨形态发生蛋白表达;空白支架组仅软骨基质骨形态发生蛋白表达。④双能X射线吸收法检测:仿生骨膜组骨矿物含量、骨密度值均高于诱导骨膜组及空白支架组(P<0.05)。⑤生物力学测试:与诱导骨膜组、空白支架组相比,仿生骨膜组最大弯曲负荷及抗弯刚度显著提高(P<0.05)。结论:体外构建的仿生骨膜能有效的修复骨缺损,其生物相容性良好,通过协同诱导、引导、传导效应,增强骨再生能力,促进缺损修复。

AIM： To construct the mimetic periosteum in vitro by the initial combination of adipose derived stromal cells （ADSCs）, recombinant human bone morphogenetic protein 2 （rhBMP-2） and chitosan/collagen protein/β tficalcium phosphate matrix, investigate the feasibility of mimetic periosteum for repairing the radial defect in rabbits, and analyze the osteogenic potential and biocompatibility of mimetic periosteum. METHODS： The experiment was carried out in the Tissue Engineering Research Center, National University of Singapore and Tianjin Medical University from May 2005 to November 2006.（1）Three-dimensional construction of mimetic periosteum was made by implantation of ADSCs into rhBMP-2 mediated chitosan/collagen protein/β tricalcium phosphate carrier, in the combination with biological gel.（2）Totally 72 New Zealand rabbits were randomly divided into 3 groups.（3）A 10-mm defect of right radius was established in rabbits, group A was transplanted with mimetic periosteum （;arrying ADSCs and rhBMP-2, group B was implanted by osteoinductive periosteum carrying rhBMP-2, and group C was implanted by chitosan/collagen protein/β tricalcium phosphate matrix as blank scaffold.（4）At 2, 4, 6, 8, 10, 12 weeks postoperatively, the bone formation and reconstruction in the radial defect area were observed. Hematoxylin-eosin staining and Masson staining were used to observe the bone tissue repairs. The expression of BMP was detected by using immunohistochemical staining; Dual energy X-ray absorptiometry was applied to assay the bone mineral content and bone mineral density. At 12 weeks postoperatively, the mechanical index and rigidity of bone structure were determined by using biomechanical test. RESULTS： All of 72 rabbits were involved in the result analysis. （1）Gross observation： Group A appeared a large amount of bone callus at 6 weeks and bone connection at 12 weeks; partly bone connection occurred in group B; bone callus grew centripetally in group C.（2）Histology： At 6 weeks, Intramembranous ossification was dominant in group A, and recovered to the normal bone structure at 12 weeks; endochondral ossification could be seen in group B, which showed primary repair at 12 weeks; played a predominant role in process of new tissue regeneration and mature bone reconstitution, defect completely healed at 12 weeks. Group C displayed lamellar bone formation. There were no inflammatory or immunological rejections in all the groups.（3）Immunohistochemistry： Hyperplastic endosteum, osteocyte of callus and medullary canal BMP were all significantly expressed in group A; both cartilage matrix and partly periosteum expressed BMP in group B; only cartilage matrix showed BMP expression. （4）Dual energy X-ray absorptiometry： Group A had statistical significance over group B and group C according to bone mineral content and bone mineral density （P 〈 0.05）. （5）Biomechanical test： Compared with group B and group C, the maximal bending load and rigidity was significantly increased in group A （P〈 0.05）. CONCLUSION： Bone defect can be healed by using mimetic periosteum, which is constructed in vitro. Enhanced osteogenic potential can be obtained with a concord pattern of osteoinductive and osteopromotive and osteoconductive effects.