血管内皮生长因子对临界下颌骨缺损纳米骨块移植的影响

The effect of vascular endothelial growth factor on the healing of nano block bone grafting in critical⁃sized mandibular defects

目的研究血管内皮生长因子(VEGF)对临界下颌骨缺损块状骨移植骨愈合的影响。方法4只比格犬,于每侧下颌骨建立4个标准化临界骨缺损。利用半口对照设计将单纯纳米羟基磷灰石珊瑚骨块(nHA/coral)和复合VEGF的nHA/coral植于缺损中,VEGF/nHA/coral组为实验组,nHA/coral组为对照组。3、8周后处死动物,通过Masson和vWF免疫组化染色观察新生血管和骨组织生长情况;通过双荧光标记观察钙化骨组织新生情况。采用SPSS 18.0统计分析软件,对vWF免疫组化染色的新生血管密度和荧光标记的钙化新生骨面积比数据进行独立样本t检验统计分析。结果3周时,Masson染色可见蓝染的胶原蛋白表达活跃,结构尚未成熟,与nHA/coral组相比,VEGF/nHA/coral组新生骨胶原纤维相对成熟整齐,局部可见骨陷窝、有钙盐沉积的骨细胞以及与伴行的血管系统。8周时,两组均可见蓝色的骨细胞和骨样组织以及成熟的骨性胶原纤维排列整齐而致密,新生骨胶原分布均明显增宽增多,有编织骨形成,红蓝相间。免疫组化染色结果显示:3周时,nHA/coral组和VEGF/nHA/coral组的新生血管密度分别为(105±31)和(146±33)个/mm2,实验组明显高于对照组(P<0.05)。8周时,两组的新生血管数量均有显著增加至(269±67)和(341±71)个/mm2,差异无统计学意义(P>0.05)。荧光标记组织形态测量结果显示:3周时,nHA/coral组和VEGF/nHA/coral组的钙化新生骨面积比分别为(0.79±0.21)%和(1.08±0.29)%,组间差异有统计学意义(P<0.05);8周时,两组的钙化新生骨面积比分别为增长至(4.25±1.13)%和(5.21±1.07)%,组间差异无统计学意义(P>0.05)。结论局部应用血管内皮生长因子可在骨愈合早期阶段显著提高血管新生和新生骨矿化程度。

Objective To study the effect of vascular endothelial growth factor on the healing of nano block bone grafting in critical⁃sized mandibular defects.Methods Four critical⁃sized defects in each side of mandible of four Beagle dogs were built.There were two groups in our study,i.e.,the VEGF/nHA/coral group acted as the experimental group,and the nHA/coral group as the control group.Pure nano⁃hydroxyapatite/coralline blocks and VEGF⁃coated blocks were implanted into the above defects in a split⁃mouth design.In three and eight weeks,the animals were euthanized,and the angiogenesis and bone regeneration in both groups were histologically observed by Masson′s trichrome and vWF staining technique.Newly formed calcified bone was evaluated by two fluorescents labeling technique.Neovascular density of vWF immunohistochemical stained blood vessels and the percentage of fluorescent marked calcified new bone area were statistically analyzed in two independent sample t⁃test in SPSS 18.0.Results At three weeks in two groups,from the Masson stained images,newly formed trabecular bone was obvious with immature collagen fibers disorderly arranged and blue stained.Compared to the newly⁃formed bone in nHA/coral group,collagen fibers in VEGF/nHA/coral group were relatively more mature and regular arranged;Osteoblasts were surrounded by osteoid bone,which evoluted into bone cells and bone lacuna;Calcium salt deposited bone cells and bone lacuna structure were blue stained.At eight weeks in both groups,blue stained bone cells and mature bone with better aligned and wider collagen fibers were observed;Mature collagen matrix secretion was significantly increased,and some woven bone formation can be easily found.In addition,immunohistochemical analysis revealed that compared to the nHA/coral samples(105±31 vessels/mm2),the VEGF/nHA/coral samples showed an increase in the neovascular density(146±33 vessels/mm2)at three weeks,with a significant difference between the two groups(P<0.05).At eight weeks,with the increasement of bone healing time,the quantity of new blood vessels in the two groups significantly increased.The neovascular density innHA/coral group and VEGF/nHA/coral group increased to 269±67 and 341±71,respectively,without significant difference(P>0.05).Fluorescence analysis results indicated that at three weeks,the percentage of calcified new bone area was(0.79±0.21)%for the nHA/coral group and(1.08±0.29)%for the VEGF/nHA/coral group,with a significant difference between two groups(P<0.05).At eight weeks,fluorescence labeled new bone in periphery and internal of scaffolds was significantly increased,and yellow tetracycline fluorescence can be easily observed in the different parts of blocks in two groups.PMBF in both groups increased to(4.25±1.13)%and(5.21±1.07)%,respectively,with no significant difference(P>0.05).Conclusion The local delivery of VEGF can significantly improve neovascularization and mineralization of newly formed bone at the early stage of block bone healing in this dog implantation model.