骨结合过程中种植体周血管时空分布的观测研究

Spatio-temporal imaging and assessment of neovascularization in implant osseointegration via micro-computed tomography

目的探讨构建一种观测小鼠股骨种植体周围骨血管的改良方法,并分析局部血管在种植体周围骨愈合过程中时空变化规律。方法对C57BL/6小鼠双侧股骨进行血管阻射剂灌注,并应用显微计算机断层扫描(Micro-CT)技术对种植体周围血管进行扫描,定量分析三维血管结构与分布的相关参数。其中左侧股骨干骺端在灌注前作种植体骨结合模型(灌注实验组),右侧股骨干骺端未行手术(空白对照组)。同步术后观察时间点1、3、7和14 d,采用组织学检测作为Micro-CT结合血管灌注改良观测方法的验证(组织学对照组)。通过空白对照组排除各时间点样本原始血管的差异,采用单因素方差分析结合Turkey′s法对灌注实验组进行各时间点之间种植体周围血管数据参数的比较。结果通过采用推出种植体的脱钙骨组织样本、筛选扫描重建参数的改良Micro-CT结合血管灌注方法,发现相较于空白对照组,灌注实验组种植体周围血管的血管总体积、血管体积分数及血管管径在术后7 d均较术后1、3和14 d有统计学差异。其中,术后7 d的血管体积(F=14.340,P_(1 d)<0.001,P_(3 d)=0.001)、血管体积分数(F=16.230,P_(1 d)<0.001,P_(3 d)<0.001)、血管管径(F=9.427,P_(1 d)=0.006,P_(3 d)=0.002)显著高于术后1、3 d;术后7 d的血管分支数量显著高于术后1 d(F=4.723,P_(1 d)=0.019);但术后14 d植体周围组织血管结构参数相较于术后7 d出现显著下降,其中血管体积分数下降了58%(F=16.230,P=0.006),血管平均管径下降了35%(F=9.427,P=0.041)。组织学对照组与灌注实验组观察结果趋势一致。结论改良Micro-CT扫描重建结合血管灌注方法为观测种植体周围血管时空分布提供一种低成本可靠的途径;种植体骨结合过程中种植体周围血管经历先增加再衰退的过程,在术后1周的高峰期可形成侧支循环围绕种植体。

Objectives To establish an evaluation model for peri-implant vasculature in the mouse femur in order to analyze spatio-temporal dynamics of vasculature around implants using a feasible and reliable method.Methods C57BL/6 mouse femurs were performed vascular perfusion with radiopaque agents and then scanned by Micro-CT with particular scanning and reconstruction parameters to image and quantify angiogenesis around implants in the distal ends.The left femurs were assigned to the perfusion experiment group with transcortical defects(Φ1 mm)for implant placement,while the right femurs were assigned to the negative control group without implant surgery.Mice were sacrificed on Day 1,3,7 and 14 post-surgery.Histological examination was performed simultaneously for comparison as the positive control group.The baseline was confirmed not significant by comparisons between varied timepoints within the negative control group.The vascular parameters in the perfusion experiment group were analyzed by using One-Way ANOVA and the Turkey′s test(P<0.05).Results We proposed a modified method by managing decalcified samples with implants pushed-out before Micro-CT scanning and setting up low-pass Gaussian filterσvalue and threshold.In contrast to the negative control group,there was significant difference between the peri-implant vasculature on Day 7 and the counterpart on Day 1 and Day 3 post-surgery.The peri-implant vasculature on Day 7 displayed higher vascular volume(F=14.340,P_(1 d)<0.001,P_(3 d)=0.001),vascular volume fraction(F=16.230,P_(1 d)<0.001,P3 d<0.001)and vessel thickness(F=9.427,P_(1 d)=0.006,P_(3 d)=0.002)compared with those on Day 1 and 3.The number of vessel branches was also larger on Day 7 compared with that on Day 1(F=4.723,P_(1 d)=0.019).Meanwhile,peri-implant vasculature decreased significantly from Day 7 to Day 14 with a reduction of 58%on the vascular volume fraction(F=16.230,P=0.006)and 35%on the average vessel thickness(F=9.427,P=0.041).The histological observation was in accordance with the outcomes above.Conclusions We proposed a feasible and low-cost Micro-CT modified method for observing and quantitating three-dimensional dynamics of angiogenesis around implant.The dynamic variation of peri-implant vasculature was observed,that is,the vasculature first increased together with vasodilated and then decreased.At its peak,the branch grew into collateral vessels around titanium implants within 7 days post-surgery.