负载重组人骨形态发生蛋白2的α型半水硫酸钙/纳米羟基磷灰石复合植骨材料的成骨性能研究

Study of osteogenic properties of recombinant human bone morphogenetic protein-2 loaded α-hemihydrate calcium sulfate/nano-hydroxyapatite combined bone materials

目的：评价负载重组人骨形态发生蛋白2（rh BMP-2）的α型半水硫酸钙/纳米羟基磷灰石（α-CSH/n HA）复合植骨材料的成骨性能。方法：制备可注射性α-CSH/n HA/rh BMP-2复合材料,取12只成年绵羊,分别经左侧椎弓根在L1-L6椎体上制作直径6mm、深15mm的6个洞形缺损,第一只绵羊的椎体缺损随机分为3组,其余羊的椎体都按此进行分组,即L2、L5为实验组,L1、L4为对照组,L3、L6为空白对照组。实验组植入α-CSH/n HA/rh BMP-2复合材料;对照组植入可注射性磷酸钙骨水泥（CPC）;空白对照组不植入任何材料。术后4、8、12周各处死4只动物,取椎体标本分别行X线及micro-CT扫描,观察缺损修复情况;行生物力学检查,测定椎体压缩强度和压缩模量;行组织学观察比较新骨生成率。结果：术后4周时,实验组压缩强度及压缩模量与CPC组的差异无统计学意义（P〉0.05）,实验组和对照组均显著性高于空白对照组（P〈0.05）;术后8周和12周时实验组和对照组均显著性高于空白对照组（P〈0.05）,实验组显著性高于对照组（P〈0.05）。影像学及组织学结果显示,术后4周时,实验组复合材料大部分已降解,密度稍低于正常骨,可见大量纤细短小、尚未塑形的新生骨小梁,边缘有大量成骨细胞环绕;对照组CPC材料呈高密度充满整个缺损,未见明显降解吸收征象,材料与骨的界限明显,缺损边缘有少量幼稚骨小梁形成;空白对照组缺损较大,边缘整齐,几乎看不到有新骨生成。术后8周时,实验组复合材料完全降解,缺损内新生骨小梁数量增多,增粗变长,且开始早期塑形;对照组材料开始部分降解,在边缘和中心降解区可见明显新骨形成,新生骨小梁数量较实验组少;空白对照组缺损修复不明显,边缘只可见少量新生骨小梁。术后12周时,实验组缺损完全被新生骨小梁充满,骨小梁趋于成熟,结构与正常骨小梁相似,密度与正常骨接近,很难与正常部位区分;对照组CPC大量降解,只残留一小团块状和零星的材料,缺损边缘修复明显,新生骨小梁长入残余材料内,将其分割成岛屿状;空白对照组缺损依然较大,呈低密度影清晰可见,新骨生成很少。结论：α-CSH/n HA/rh BMP-2复合材料在绵羊体内具有良好的成骨活性,是较好的微创植骨材料。

Objectives： To evaluate osteogenic properties of recombinant human bone morphogenetic protein-2（rh BMP-2） loaded novel α-hemihydrate calcium sulfate/nano-hydroxyapatite（α-CSH/n HA） combined bone materials.Methods： Injectable α-CSH/n HA/rh BMP-2 composites had been developed first,then 6 hole defects with 6.0mm in diameter and 15.0mm in depth were experimentally created in left pedicles of L1-L6 lumbar vertebrae in 12 adult sheep.Defects of the first sheep were randomly divided into three groups,and which was the standard of subsequent 11 sheep.The experimental group（L2,L5） was filled with α-CSH/n HA/rh-BMP-2 composites,control group（L1,L4） with injectable calcium phosphate cement（CPC）,blank group（L3,L6）with none material.After 4,8 and 12 weeks,four animals were sacrificed and 24 vertebral specimens were taken respectively,then the followings were made respectively： X-ray and micro-CT imaging for observation of repairing,and biomechanical examination for testing compression strength and compression modulus,as well as histological evaluation by comparing new bone formation rate.Results： Biomechanical results illustrated that after 4 weeks,the difference of compression strength and compression modulus between experimental group and control group was not significant（P〉0.05）,and both significantly higher than the blank group（P〈0.05）; after8 or 12 weeks,they were also much higher than the blank group（P〈0.05）,the compression strength and compression modulus of experimental group were higher than those of control group（P〈0.05）.Radiographic and histological results indicated that after 4 weeks,the vast majority composites of the experimental group had yet degraded,the density was slightly lower than that of normal bone,and a large number of short and notyet-shaping new bone trabeculae were found surrounded by many osteoblasts; the defect was full of high density CPC material in control group,with no obvious sign of degradation,the bone-material boundary was obvious,and a small amount of immature bone formatted at the edge of the defect; while the defect of blank group was large with neat edge,where the new bone formation was barely.At 8 weeks after operation,the degradation of combined materials in experimental was apparently complete,the new bone trabeculae within the defect became larger,thicker and longer,and started early shaping; in control group,CPC began to degrade partially,new bone formation was shown at the edge and central degradation area,which was less than that of the experimental group; on the contrary,the defect healing of blank group was not obvious,there was few new bone trabeculae at the edge.After 12 weeks,the defect of experimental group was completely filled with maturing new bone trabeculae,in which the structure and density were almost as the same as normal bone trabeculae,it was difficult to distinguish the defect area from normal site; while CPC was substantially degraded in control group,remaining only a small lump and some sporadic materials,and defect restoration was obvious at the edge,new bone trabeculae grew into the residual material,which were divided into series of island-like pieces; defects in blank group were still large,presented as clearly visible low density,with very little new bone formation.Conclusions： The α-CSH/ n HA/rh BMP-2 composite is a promising bone repair material for minimally invasive surgery,which performs outstanding osteogenic properties in vertebral defect of sheep.