中国修复重建外科杂志

中国修复重建外科杂志

“手风琴”技术在牵张成骨区新生骨矿化中的作用

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目的 探讨“手风琴”技术对大鼠牵张区骨再生的作用、刺激时机以及可能机制。 方法 取 12 周龄雄性 SD 大鼠 54 只,制备右侧胫骨牵张成骨模型。术后经 5 d 潜伏期、7 d 牵张期后进入为期 6 周的矿化期。根据在矿化期实施“手风琴”操作的时间不同,将大鼠随机分为 4 组:对照组(n=18)为观察组,不行“手风琴”操作;早期组(n=18)、中期组(n=12)及晚期组(n=6)分别于矿化第 1、3、5 周行“手风琴”操作,操作时间为 7 d,其中 3.5 d 压缩、3.5 d 牵张。对照组和早期组分别于矿化第 2、4、6 周结束时,中期组于矿化第 4、6 周结束时,晚期组于矿化第 6 周结束时,各取 6 只大鼠处死,取双侧胫骨进行相关观测。矿化期间每周对各组大鼠右侧胫骨摄正侧位 X 线片,观察骨再生与矿化进展;各组每次取材时对右侧胫骨行 Micro-CT 扫描三维重建观察,评估牵张区新生骨结构改变,并计算矿化第 6 周各组 158~211、211~1 000 及 158~1 000 阈值范围的骨体积(bone volume,BV)和组织体积(tissue volume,TV),计算两者比值 BV/TV,同时计算骨密度(bone mineral density,BMD)。矿化第 6 周,对所取胫骨行四点弯曲生物力学检测最大压力、弹性模量、折断能量;行 Von Kossa、番红 O、HE 染色及成骨相关转录因子(osterix,OSX)、骨钙蛋白(osteocalcin,OCN)、VEGF 免疫组织化学染色观察。 结果 X 线片显示牵张区骨痂在矿化中期组行“手风琴”刺激后矿化速度最快。Micro-CT 三维重建可见中期组骨重建最快,牵张后 6 周皮质获得连续。矿化第 6 周,中期组 BMD 及 158~1 000、211~1 000 阈值范围的 BV/TV 均高于其余各组。生物力学检测示,中期组胫骨标本的最大压力、弹性模量、折断能量均显著高于其余 3 组(P<0.05)。组织学染色示中期组骨重建速度最快,且至牵张后第 6 周髓腔基本获得再通。免疫组织化学染色结果进一步提示,中期组新生骨组织内成骨指标(OCN、OSX)及成血管指标(VEGF)随着骨重建的进程明显升高后又恢复正常。 结论 “手风琴”技术有利于牵张成骨区新骨形成,其中矿化中期进行“手风琴”操作能更快速、有效地促进大鼠骨矿化与重建。

Objective To investigate the effect, right timing, and mechanism of " accordion” technique on bone regeneration in rat distraction osteogenesis model. Methods Fifty-four 12-week-old male Sprague Dawley rats underwent right tibial distraction osteogenesis procedure. After a 5-day latency, the distraction was performed for 7 days followed by 6-week consolidation. All animals were randomly divided into 4 groups based on different periods of " accordion” maneuvers in consolidation phase: control group (n=18) with no manipulation, and three experimental groups including early-phase group (n=18), mid-phase group (n=12), and late-phase group (n=6) with " accordion” maneuvers applied at 1, 3, and 5 weeks, respectively. The duration of the " accordion” maneuver was 7 days consisting of a 3.5-day compression and 3.5-day distraction. Rats in control group and early-phase group were sacrificed at 2, 4, and 6 weeks of the consolidation phase; rats in mid-phase group were sacrificed at 4 and 6 weeks of the consolidation phase; and rats in late-phase group were sacrificed at 6 weeks of the consolidation phase. Bilateral tibias from 6 rats in each group at each time point were obtained. Callus formation was monitored by X-ray radiography every week; new bone was reconstructed by Micro-CT three-dimensional reconstruction. The change of bone structure was evaluated, and parameters containing bone volume (BV)/tissue volume (TV) ratio (BV/TV) and bone mineral density (BMD) in three thresholds (158-211, 211-1 000, 158-1 000) were recorded and calculated at 6 weeks. Mechanical test consisting of ultimate load, modulus of elasticity, and energy to failure was performed. Histological analysis, such as Von Kossa staining, Safranin O staining, and HE staining, was done. Immunohistochemical staining using markers of osterix (OSX), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) was analyzed. Results Images of X-ray showed that callus formation increased significantly in the mid-phase group. Micro-CT three-dimensional reconstruction demonstrated the mid-phase group owned fastest reconstructed speed among 4 groups, the cortical bone was continual at 6 weeks. At 6 weeks, the BMD and BV/TV in thresholds 158-1 000 and 211-1 000 in mid-phase group were higher than those in other groups. The results of mechanical test showed that ultimate load, modulus of elasticity, and energy to failure in mid-phase group were significantly higher than those in other groups (P<0.05). Histological testing showed that the continuity of bone marrow cavity in mid-phase group was evident at 6 weeks after distraction. Immunohistochemical analyses confirmed the expression levels of osteogenesis (OCN, OSX) and angiogenesis (VEGF) elevated remarkably and then returned to normal in mid-phase group. Conclusion The " accordion” technique is beneficial for new callus formation in distraction area. Applying the maneuver during the middle phase of the consolidation period was effective to accelerate new bone formation in rat distraction osteogenesis model.

关键词: “手风琴”技术; 牵张成骨; 骨矿化; 大鼠

Key words: " Accordion” technique; distraction osteogenesis; bone consolidation; rat

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