异种脱蛋白松质骨支架构建组织工程骨用于脊柱横突间融合(英文)

Construction of tissue-engineered bone using a xenogenic deproteinized bone scaffold for spinal intertransverse fusion

背景:异种脱蛋白松质骨来源广泛且具有独特的生物学特性,若能解决其免疫原问题,将有望利用异种脱蛋白松质骨开发出一种新型植骨材料。目的:探讨异种脱蛋白松质骨作为骨组织工程支架的性能,及其在山羊脊柱横突间融合中的作用。设计、时间及地点:细胞-支架学体内实验,于2008-02/10在黑龙江省纤维化生物治疗重点实验室完成。材料:6～8月龄健康雄性山羊12只,由牡丹江医学院动物中心提供。方法:取成年猪股骨远端松质部分,通过理化方法制成脱蛋白松质骨支架,对其形态结构、组成成分、生物力学特性以及材料对种子细胞生物学行为的影响进行检测分析。羊髂骨穿刺取骨髓,梯度密度离心法分离获得第3代骨髓间充质干细胞。将支架材料复合一定量的自体骨髓间充质干细胞、重组人骨形成蛋白2构建组织工程骨。12只羊建立L3、4双侧横突间植骨模型,修复组于左侧植入组织工程骨,对照组于右侧植入相同体积的自体髂骨。主要观察指标:分别于植入后4,8,12周行X射线片和组织学观察对比分析。结果:采用脱蛋白处理后的松质骨可见大小不等、相互交通、开放孔隙的网架结构,孔径200～500μm,孔隙率60%左右,其无机成分为羟基磷灰石,有机成分为胶原,力学性能保存良好,细胞相容性良好。X射线片结果显示:植入第4周,横突桥接处部分区域模糊,以内侧明显,此时修复组材料密度略低于对照组;第8周上下桥接部间隙变小,大量连续骨痂生成;12周后完全融合,两组材料密度接近。移植区组织学观察结果显示:修复组植入后第4周以多点方式形成新骨,第8周岛状生成的骨组织贯穿整个移植材料,12周编织骨交错排列,髓腔形成,成骨活性接近自体髂骨移植;对照组植入后4周有较多新骨形成,8周时出现大量胶原纤维,周边成骨明显,至12周纤维组织减少,成骨活跃。结论:以异种脱蛋白松质骨为支架材料,复合骨髓间充质干细胞、重组人骨形成蛋白2构建组织工程骨,体外X射线衍射分析及力学测试与体内成骨实验均证明其具有良好的组织相容性和较强的成骨能力,是一种良好的组织工程骨支架材料。

BACKGROUND： Xenogenic deproteinized bone is generally collected and has unique biological properties; however, its immunogen hopes be resolved so as to discover a new type of bone graft material. OBJECTIVE： To explore the performance of xenogenic deproteinized bone scaffold for tissue-engineered bone and the effect on spinal intertransverse fusion in goats. DESIGN, TIME AND SETTING： An in vivo cell-scaffold study was performed at the Key Laboratory of Fibrotic Biotherapy of Heilongjiang Province between February and October 2008. MATERIALS： Twelve healthy male goats aged 6 8 months were provided by Animal Center of Mudanjiang Medical College. METHODS： Cancellous bone at distal femur of adult swine was obtained to prepare xenogenic deproteinized bone scaffold using physical and chemical methods. Effects of the scaffold on morphology, structure, component, biomechanical property, and biological behavior of seed cells were detected and analyzed. Bone marrow was extracted from ilium of goat and gradient-centrifuged to obtain the third-passaged bone marrow mesenchyme stem cells （BMSCs）. A certain quantity of autologous BMSCs and recombinant human bone morphogenetic protein 2 （rhBMP-2） were plated onto the scaffold to achieve tissue-engineeried bone. Models of bilateral L3,4 intertransverse bone graft were established on 12 goats. Tissue-engineeried bone was implanted into the left side in a repairing group, while the same volume of autologous ilium was implanted into the right side in a control group. MAIN OUTCOME MEASURES： X-ray examination and histological detection were performed at 4, 8, and 12 weeks after implantation. RESULTS： Deproteinization-treated cancellous bone exhibited a spatial grid structure of variously sized, crossing and opening pores, composing hydroxyapatite and collagen. The pore diameter was 200 500 pro, and porosity was about 60%. Mechanical property and cell compatibility were well. X-ray examination demonstrated that at 4 weeks after implantation, some or even lateral intertransverse bridging regions were unclear, and material density in the repairing group was lower than control group; at 8 weeks after implantation, interspace between up and down bridging was shrunk, and a great quantity of calluses were successively formed; at 12 weeks after implantation, complete confluence was observed, and the density in the repairing group was closed to that in the control group. Histological detection indicated that at 4 weeks after implantation, new bone was multidrop-formed in the repairing group; at 8 weeks after implantation, bone tissue like the islet shape was passed through the whole implanted materials; at 12 weeks after implantation, woven bones arranged across, medullary cavity was formed, and osteogenic activity in the repairing group was closed to that in the control group. On the other hand, at 4 weeks after implantation, a large quantity of new bones were formed in the control group; at 8 weeks after implantation, a great quantity of collagen fibers were observed, and osteogenesis was clear in marginal region; at 12 weeks after implantation, fibrous tissues were reduced, and osteogenesis was active. CONCLUSION： BMSCs and rhBMP-2 incubated on xenogenic deproteinized cancellous bone is an ideal tissue-engineeried bone scaffold, characterizing by an excellent histocompatibility and a strong osteogenesis, based on in vitro X-ray diffraction analysis, mechanical test, and in vivo osteogenesis experiment.