重组人BMP-2缓释作用于基质血管成分细胞促进大鼠脊柱融合的实验研究

发布时间：2018-05-29 12:59

本文选题：基质血管成分细胞 + 重组人BMP-　；参考：《中国修复重建外科杂志》2017年07期

【摘要】：目的将重组人BMP-2(recombinant human BMP-2,rh BMP-2)缓释作用于新鲜分离的大鼠基质血管成分细胞(stromal vascular fraction cells,SVFs),移植到大鼠腰椎横突后外侧,观察其促进脊柱融合的效果。方法取健康4月龄SD大鼠双侧腹股沟区皮下脂肪,采用Ⅰ型胶原酶消化法提取SVFs。通过仿生磷灰石涂层方法,将rh BMP-2与β-磷酸三钙(β-tricalcium phosphate,β-TCP)通过共价键结合制成缓释载体,通过BCA法测定其缓释效应。将32只大鼠随机分成4组,每组8只。采用L_4、L_5横突去皮质化方法制备大鼠腰椎后外侧融合模型,分别于A、B、C、D组植入PBS+脱钙骨基质(decalcified bone matrix,DBX)、rh BMP-2/β-TCP缓释载体+DBX、SVFs+DBX和SVFs+rh BMP-2/β-TCP缓释载体+DBX。术后8周获取腰椎融合标本,进行手法判断和X线片观察评价脊柱融合情况,以及micro-CT评价脊柱融合情况,并分析骨密度(bone mineral density,BMD)及骨体积分数;行HE染色、Masson三色组织学染色,以及免疫组织化学染色检测骨钙蛋白(osteocalcin,OCN)。结果 2周时rh BMP-2/β-TCP缓释载体的rh BMP-2体外累计释放量为40%左右,起到缓慢释放效果;而未经仿生磷灰石涂层的对照组2周时几乎停止释放rh BMP-2。手法判断、X线片和micro-CT评估一致,显示D组成骨能力最强,达100%融合(8/8),之后依次为B组(75%、6/8)、C组(37.5%、3/8)、A组(12.5%、1/8)。Micro-CT分析示,D组BMD和骨体积分数均显著高于其余3组,B组显著高于A、C组,差异均有统计学意义(P0.05)。HE、Masson三色染色和OCN免疫组织化学染色示,D组有大量软骨细胞连接,伴有骨基质沉积、有活跃的成骨过程,类似长骨的矿化过程;B组的骨形成相对D组较弱;A、C组缺乏有效的新骨形成。结论新鲜分离的SVFs在rh BMP-2缓释作用下,显示出良好的骨生成能力,可有效促进脊柱融合,为临床应用提供理论基础。
[Abstract]:Objective to investigate the effects of sustained release of recombinant human BMP-2(recombinant human BMP-2rh BMP-2 on newly isolated rat stromal vascular fraction cells (SVFsS) and to observe the effect of promoting spinal fusion by transplanting the recombinant human BMP-2rh BMP-2 to the posterolateral side of the lumbar vertebrae. Methods the subcutaneous fat of bilateral inguinal region of 4 month old SD rats was extracted by type I collagenase digestion. Rh BMP-2 and 尾 -tricalcium phosphate (尾 -TCPA) were covalently bonded to prepare slow-release carrier by bionic apatite coating method. The slow-release effect of rh BMP-2 was determined by BCA method. Thirty-two rats were randomly divided into 4 groups with 8 rats in each group. The posterolateral fusion model of lumbar vertebrae in rats was established by decorifying L4 / L5 transverse process. PBS decalcified bone BMP-2/ 尾 -TCP DBX and SVFs BMP-2/ 尾 -TCP sustained-release carriers were implanted into PBS decalcified bone matrix group. Lumbar fusion specimens were obtained 8 weeks after operation. The spinal fusion was evaluated by manipulation, X-ray observation and micro-CT. Bone mineral density (BMD) and bone volume fraction (BMD) were analyzed. Osteocalcinin OCNA was detected by immunohistochemical staining. Results the accumulative release of rh BMP-2/ 尾 -TCP in vitro was about 40% at 2 weeks, which showed a slow release effect, while the control group without bionic apatite coating almost stopped releasing rh BMP-2 at 2 weeks. The results showed that the osteogenic ability of group D was the highest, with 100% fusion rate of 8 / 8%, followed by group B (group B) with 37.5% / 8% trochanter than group C (n = 37.5%). Micro-CT analysis showed that the BMD and bone volume fraction in group D were significantly higher than those in group B and group A (P < 0.05), and the bone volume fraction in group B was significantly higher than that in group A (P < 0.05). The bone mass fraction in group D was significantly higher than that in group A (P < 0.05), and the bone volume fraction in group B was significantly higher than that in group A. The differences were statistically significant (P 0.05) and OCN immunohistochemical staining showed that there were a large number of chondrocyte junctions, bone matrix deposition and active osteogenesis in group D. The formation of bone in group B was less effective than that in group D in mineralization process similar to long bone. Conclusion the newly isolated SVFs showed good bone-forming ability under the sustained release of Rh BMP-2, which could effectively promote spinal fusion and provide theoretical basis for clinical application.
【作者单位】：上海健康医学院附属周浦医院骨科;上海中医药大学附属岳阳中西医结合医院骨伤科;
【基金】：上海市浦东新区卫生系统学科带头人培养计划(PWRd2015-02) 上海健康医学院种子基金项目(HMSF-16-21-025) 上海市卫生和计划生育委员会科研课题面上项目(201440387) 上海市医学重点专科建设项目(ZK2015A14)~~
【分类号】：R68

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