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

Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model

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(P<0.05)。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。

Objective To observe the effect ofstromal vascular fraction cells （SVFs） from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 （rhBMP-2） in promoting the lumbar fusion in rat model. Methods SVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β- tricalcium phosphate （β-TCP） by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix （DBX） scaffold＋PBS, DBX scaffold＋rhBMP-2/β-TCP sustained release carrier, DBX scaffold＋SVFs, and DBX scaffold＋rhBMP-2/β-TCP sustained release carrier＋SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density （BMD） and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson＇s trichrome staining, osteocalcin （OCN） was detected by immunohistochemical staining. Results The cumulative release amount ofrhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation （100%, 8/8）, followed by group B （75%, 6/8）, group C （37.5%, 3/8）, and group A （12.5%, 1/8）. Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C （P〈0.05）, and in group B than groups A and C （P〈0.05）. HE staining, Masson＇s trichrome staining, and immunohistochemistry staining for OCN staining exhibited a large number of cartilage cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and there was no effective new bone formation in groups A and C. Conclusion The combination of sustained release of rhBMP-2 and freshly SVFs can significantly promote spinal fusion in rat model, providing a theoretical basis for further clinical applications.