不同培养时间β-磷酸三钙／骨髓基质干细胞组织工程骨在兔脊柱后外侧融合中的作用研究

Effect of different incubation durations of β-TCP/BMSCs tissue engineering bone on the posterolateral spinal fusion in rabbits

目的探讨β-磷酸三钙／骨髓基质干细胞（β—TCP／BMSCs）组织工程骨培养不同时间对兔脊柱后外侧融合的作用。方法分离培养BMSCs并经体外成骨诱导后接种至β-TCP上，分别培养1、3、7d后植入新西兰大白兔体内，行脊柱后外侧融合。利用噻唑蓝、扫描电镜、荧光染色等方法观察体外细胞活力和生长情况；术后12周处死动物，行X线、显微-CT、组织学及组织形态计量学检测，观察新生骨形成和融合情况。结果1、3、7d组细胞活力分别为（0．19±0．02）A／g、（0．48±0．06）A／g、（0．61±0．05）A／g，3组间两两比较差异均有统计学意义（F=175．570，P=0．000）。扫描电镜及荧光染色显示细胞生长良好；术后12周1d组X线评分、脊椎融合率、BMC、BV／TV、新生骨面积百分比均低于于3、7d组，差异均有统计学意义（P〈0．05），但3、7d组比较差异均无统计学意义（P〉0．05）。结论β-TCP／BMSCs组织工程骨培养时间明显影响兔脊柱后外侧融合效果，但融合效果并不随培养时间延长而明显提高，提示培养时间存在“临界值”。

Objective To investigate appropriate incubation time of bone marrow stem cells （BMSCs） combined with β-triealeium phosphate （β-TCP） for the posterolateral spinal fusion in rabbits. Methods The rabbit BMSCs were isolated and cultured to induce osteoblasts in vitro. The same amounts of osteoblasts were seeded onto β-TCP for static incubation of 1, 3 and 7 days respectively before implantation into New Zealand rabbits for the posterolateral spinal fusion. The cell viability was observed by MTT, scanning electron microscopy and fluorescent staining. The rabbits were sacrificed 12 weeks postoperation for radio- logical, histological and histomorphometry observations of new bone formation and lumbar fusion. Results The cell viability was 0. 19 ± 0.02 A/g for incubation of one day, 0.48 ± 0. 06 A/g for incubation of 3 days and 0. 61 ± 0. 05 A/g for 7 days, with a significant between-group difference （ F = 175. 570, P = 0. 000）. Scanning electron microscopy and fluorescent staining showed fine growth of the cells. At 12 weeks the X-ray score, spinal fusion rate, bone mineral content （BMC）, bone volume fraction （BV/TV） value, percentage of new bone formation for incubation of one day were all significantly lower than those for incubation of 3 and 7 days （ P 〈 0. 05 ）, but there was no significant difference between incubation of 3 days and incubation of 7 days （ P 〉 0. 05 ）. Conclusions The posterolateral spinal fusion is significantly affected by the incubation time of β-TCP/BMSCs, but the incubation time is not proportional to the fusion effect, suggesting that the incubation time may have a critical value.