活体顺序性荧光标记技术在实验性骨折愈合研究中的应用

Application of sequential fluorescent labeling in vivo in the study on experimental fracture healing

目的介绍实验性骨折愈合研究中活体顺序性荧光标记的技术与方法。方法以成年兔胫骨骨折外固定为模型,术后分别以Tetracydin(TC,黄色,25mg/kg)、Calcein(CG,绿色,10mg/kg)、Xylenal(XO,桔色,90mg/kg)、Alizarin-complen(AE,红色,30mg/kg)四种荧光染料于兔颈部皮下定期注射;并于术后第3、6、12及24周活杀动物,解剖出胫骨,梯度乙醇脱水、脱脂,甲基丙烯酸甲脂包埋,切片厚度40～100μm,打磨,荧光显微镜下观察、摄像;同时通过组织学观察、X线摄片了解骨愈合速度与质量。结果顺序性荧光标记显示,加压组中钙化的内、外骨痂均于术后第2周出现于截骨处的远、近断端,但尚未形成骨性连接。亦无骨皮质的改建。外骨痂增生停止于术后12周,且骨皮质的改建至12周已渐减少,至术后24周已基本完成;对照组内骨痂于术后第3周出现于截骨处远、近断端,无钙化的外骨痂形成,亦无骨皮质的改建;外骨痂增生停止于术后12周,骨皮质改建至24周尚未完成,其结果与组织学观察、X线摄片具有一致性。结论活体顺序性荧光标记技术可以从细胞水平判断新骨的开始时间、骨生长速度及生长方向,并能节约实验动物数量,是实验性骨折愈合研究中较为理想的研究手段。

Objective To introduce a method of sequential fluorescent labeling in vivo in the study on experimental fracture healing. Methods The established model of adult rabbit tibia fracture treated with external fixation was employed. The therapeutic effects were observed with histological examination, sequencing florescent labeling, X-ray test, at the 3th, 6th, 12th, 24th week postoperatively. Fluorescent dyes and their dosages were as follows: a. Tetracyclin (yellow) 25?mg/kg; b.Calcein (green) 10?mg/kg; c.Xylenal (orange) 90?mg/kg; d.Alizarin-complen (red) 30?mg/kg. They were all injected subcutaneously at the neck. All the rabbits at each time phase were killed to obtain the tibia. The tibia was dehydrated and defatted with gradient spirit, embedded with methyl methacrylate, sectioned and ground to a thickness of 40-100?μm and samples were observed under the fluorescent microscope, and picture were taken. Results 2 weeks postoperatively, calcified internal and external callus appeared on the distal and proximal ends of the fracture. However, there was neither bony connection nor restructuring of bone cortex. The proliferation of external callus stopped at 12 weeks postoperatively. Meanwhile, the restructuring of bone cortex decreased. 24 weeks postoperatively, the bony connection completed in compressed groups, and 3 weeks after the operation, internal callus appeared on both ends of the fracture. There was formation of calcified external callus nor restructuring of bone cortex. The proliferation of external callus stopped at 12 weeks postoperatively neither. The restructuring of bone cortex did not complete 24 weeks postoperatively in control groups. Additional, the sequential fluorescent labeling results were consistent with that of histological examination and X-ray test. Conclusions On the basis of this study, we believe that sequential fluorescent labeling can determine the beginning, speed and direction of repairing bone from cellular level, meanwhile this method can reduce experimental animal amount.