目的:连续观察骨缺损愈合早期血管样组织和骨样组织的变化过程,初步了解局部施用甲磺酸去铁胺(deferoxamine mesylate,DFO)对血管化及骨再生的作用,验证DFO维持缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)活性的能力。方法:构建30只6~8周龄雄性SD大鼠颅骨缺损模型,随机分为DFO实验组和生理盐水对照组,于颅骨缺损后第4天分别局部注射200 μmol/L DFO溶液300 μL或生理盐水300 μL,并于缺损后第5、7、10、14和28天,每组每次处死3只大鼠。采用CD31免疫组织化学染色检测血管数量,采用HE染色、Masson染色观察成骨及矿化情况,以HIF-1α免疫组化染色检测HIF-1α蛋白相对表达量。结果:术后第5、7、10、14和28天,实验组血管数量(个)分别为:30.40±12.15、62.00±17.87、73.43±15.63、40.00±7.84和48.71±11.64,对照组血管数量(个)分别为:18.75±6.63、19.13±2.80、51.35±16.21、27.18±7.32和30.88±13.43,各时间点实验组血管数量均显著多于对照组(P<0.05);术后第14和28天,实验组新生骨组织较多,新生骨组织矿化百分比分别为(27.73±5.93)%和(46.53±3.66)%,对照组分别为(11.99±2.02)%和(31.98±4.22)%,这两个时间点实验组矿化百分比显著高于对照组(P<0.001);术后第5、7、10、14和28天,实验组相较对照组的HIF-1α蛋白相对表达量为2.86±0.48、1.32±0.26、1.32±0.32、1.28±0.38、1.05±0.34,仅术后第5天两组间差异有统计学意义(P<0.01)。结论:在骨缺损局部使用DFO可促进血管化及骨再生,可短暂维持HIF-1α蛋白活性。

Objective: To investigate the effect of local administration of deferoxamine mesylate (DFO) on vascularization and osteogenesis and its ability to maintain the activity of hypoxia inducible factor-1α (HIF-1α), by constantly observing early changes of vessel-like structures and bone tissues during bone defects healing. Methods: Skull critical bone defect models were constructed on a total of thirty male SD rats (6-8 weeks old). The rats were randomly divided into experimental group (DFO group) or control group (normal saline group). 300 μL 200 μmol/L DFO solution or normal saline was locally injected on the 4th day after the defect was made. On the 5th, 7th, 10th, 14th, and 28th days after surgery, three rats in each group were sacrificed respectively. HE staining and Masson staining were performed to observe new bone formation and mineralization. HIF-1α immunohistochemistry staining was performed to examine relative expression of protein. Qualitative analysis and comparation were performed by t-tests on relative expression of HIF-1α, numbers of blood vessels and percentages of mineralization tissues of new bone areas. Results: On the 5th, 7th, 10th, 14th and 28th days after surgery, the average numbers of blood vessels were 30.40±12.15, 62.00±17.87, 73.43±15.63, 40.00±7.84, 48.71±11.64 in the DFO group, and 18.75±6.63, 19.13±2.80, 51.35±16.21, 27.18±7.32, 30.88±13.43 in the control group. The number of blood vessels in the DFO group was significantly higher than that of the control group at each time point (P<0.05). The mass of new bone in the DFO group was higher than that in the control group on the 14th and 28th days after surgery. The percentage of mineralization tissues of new bone area on the 14th and 28th days after injection were (27.73±5.93)% and (46.53±3.66)% in the DFO group, and (11.99±2.02)% and (31.98±4.22)% in the control group. The percentage of mineralization tissues in the DFO group was significantly higher than that of the control group at each time point (P<0.001). The relative expression of HIF-1α in the DFO group compared with the control group was 2.86±0.48, 1.32±0.26, 1.32±0.32, 1.28±0.38 and 1.05±0.34 on the 5th, 7th, 10th, 14th and 28th days, with significant expression difference on the 5th day (P<0.01). Conclusion: Use of DFO in bone defects promotes vascularization and osteogenesis in the defect area, and maintains the protein activity of HIF-1α temporarily.