异种脱蛋白松质骨为载体构建组织工程骨用于脊柱横突间融合的实验观察

SPINAL FUSION OF LUMBAR INTERTRANSVERSE PROCESS BY USING TISSUE ENGINEERED BONE WITH XENOGENEIC DEPROTEINIZED CANCELLOUS BONE AS SCAFFOLD

目的探讨异种脱蛋白松质骨（deproteinization bone，DPB）作为骨组织工程载体的性能及其用于山羊脊柱横突间融合的作用。方法取成年猪股骨远端松质骨通过理化方法制成DPB载体，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。将载体材料复合一定量的自体骨髓间充质干细胞（mesenchymal stem cells，MSCs）重组人骨形成蛋白2（recombinant human bone morphogenetic protein 2，rhBMP-2）构建组织工程骨。取6～8月龄雄性山羊12只，制成L3-4双侧横突间植骨模型，左侧植入组织工程骨为A组，右侧植入等体积自体髂骨作对照为B组。分别于4、8及12周行X线片和组织学观察并对比分析。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，有良好的细胞相容性。两组移植后不同时间点X线表现：A组植入横突间第4周与横突桥接处部分区域模糊，以内侧明显；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。早期A组密度略低于B组，12周后基本相同。移植区组织学观察：A组植入后第4周以多点方式形成新骨，第8周岛状生长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。B组，4周时有较多新骨形成；8周时出现大量胶原纤维，周边成骨明显；12周时，纤维组织减少，成骨活跃。结论异种DPB是一种良好的组织工程骨载体材料，可为再血管化和成骨细胞的分化提供一个稳定的环境。

Objective To study the properties of the xenogeneic deproteinized cancellous bone used as a scaffold in the bone tissue engineering and its application to the spinal fusion of the lumbar intertransverse process in a goat. Methods The deproteinized bone was derived from an adult pig＇s femoral cancellous bone through the physical and chemical treatments. Its morphological features, constituting components, and biomechanical properties were examined by the scanning electron microscopy, X-ray diffraction analysis, and mechanical experimental instrument. The cell-material complex was observed under the inverted phase contrast microscope to evaluate the adhesion and the growth of the osteoblasts. The experimental model of the spinal fusion of the lumbar intertransverse process was produced in 12 male goats aged 6-8 months, which were divided into two groups. In Group A, the tissue engineered bone constructed by the xenogeneic deproteinized cancellous bone, the recombinant human bone morphogenetic protein 2, and the mesenchymal stem cells was used for the spinal fusion; however, in Group B the auto-ilium was used. The samples were harvested at 4, 8 and 12 weeks postoperatively, and a series of examinations were performed, including the radiography and the histomorphological assay Results The deproteinized cancellous bone had a natural pore network system, with an aperture ranging in size from 200 to 500μm, containing a main organic material of collagen and the inorganic material of hydroxyapatite. So, the deproteinized cancellous bone had a good mechanical strength and a good histoeompatibility. In Group A, the X-ray examination at different timepoints postoperatively showed that at 4 weeks, the bridging areas of all the fusion sites were not clear, especially on the internal side; at 8 weeks, the upper and lower bridged parts had a narrowed gap, with formation of much continuous bony callus; at 12 weeks, a complete fusion occurred. In the early stage, the material density was slightly lower in Group A than in Group B, but at 12 weeks the density was almost the same in both the groups. Histological examination in the transplant area showed that at 4 weeks in Group A there was a new bone formation in a multipoint way; at 8 weeks, a ＂sandwich-shaped＂ new bone was crossed with the transplanting materials; and at 12 weeks, a medullary cavity was remodeled and a new cancellous bone was formed. The osteogenic process of the tissue engineered bone constructed by the xenogeneic deproteinized cancellous bone scaffold was almost the same as the auto-ilium osteogenesis. Conclusion The xenogeneic deproteinized cancellous bone is a good material in the bone tissue engineering, which can be used as an osteogenesis scaffold and provide a stable environment for revascularization and osteoblastic differentiation.