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中华口腔医学研究杂志(电子版) ›› 2024, Vol. 18 ›› Issue (01) : 22 -29. doi: 10.3877/cma.j.issn.1674-1366.2024.01.004

论著

骨结合过程中种植体周血管时空分布的观测研究
高思勇, 郭彦君, 陈晖璐, 邓飞龙, 宫苹, 王天璐()   
  1. 中山大学附属口腔医院,光华口腔医学院,广东省口腔医学重点实验室,广东省口腔疾病临床医学研究中心,广州 510055
    四川大学华西口腔医院,口腔疾病研究国家重点实验室,国家口腔疾病临床研究中心,成都 610041
    南方医科大学口腔医院(口腔医学院)牙周种植科,广州 510280
  • 收稿日期:2023-02-15 出版日期:2024-02-01
  • 通信作者: 王天璐

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

Siyong Gao, Yanjun Guo, Huilu Chen, Feilong Deng, Ping Gong, Tianlu Wang()   

  1. Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangdong Provincial Clinical Research Center of Oral Diseases, Guangzhou 510055, China
    State Key Laboratory of Oral Disease & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
    Department of Periodontics and Implantology, Stomatological Hospital, School of Stomatology, Southern Medical University, Guangzhou 510280, China
  • Received:2023-02-15 Published:2024-02-01
  • Corresponding author: Tianlu Wang
  • Supported by:
    National Natural Science Foundation of China(81571008); Guangdong Basic and Applied Basic Research Foundation(2019A1515110024); Science and Technology Planning Project of Guangzhou(2023A04J2163)
引用本文:

高思勇, 郭彦君, 陈晖璐, 邓飞龙, 宫苹, 王天璐. 骨结合过程中种植体周血管时空分布的观测研究[J/OL]. 中华口腔医学研究杂志(电子版), 2024, 18(01): 22-29.

Siyong Gao, Yanjun Guo, Huilu Chen, Feilong Deng, Ping Gong, Tianlu Wang. Spatio-temporal imaging and assessment of neovascularization in implant osseointegration via micro-computed tomography[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2024, 18(01): 22-29.

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,P1 d<0.001,P3 d = 0.001)、血管体积分数(F = 16.230,P1 d<0.001,P3 d<0.001)、血管管径(F = 9.427,P1 d = 0.006,P3 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,P1 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 filter σvalue 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, P1 d<0.001, P3 d = 0.001), vascular volume fraction (F = 16.230, P1 d<0.001, P3 d<0.001) and vessel thickness (F = 9.427, P1 d = 0.006, P3 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, P1 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.

图1 C57BL/6野生型小鼠股骨干骺端钛棒植入 A:钛棒植入手术;B:植入手术后钛棒在股骨中位置示意图(粉色为钛棒与骨接触面积)。
图2 C57BL/6野生型小鼠血管灌注准备与取样结果 A:血管灌注注射泵及Microfil®阻射剂(黄色);B:灌注后C57BL/6野生型小鼠股骨及周围软组织取样,可观察到钛棒位置及阻射剂分布。
图3 显微计算机断层扫描(Micro-CT)显示小鼠股骨(远中1/2段)内主要血管分布 股骨远中段可依次区分为骨干、干骨骺、骨骺3段,其中干骨骺段为本实验钛种植体植入区域。
图4 骨内钛种植体金属伪影对显微计算机断层扫描(Micro-CT)成像小鼠股骨种植体周围血管结构的结果产生干扰 A:含钛种植体的不脱钙股骨标本扫描后成像;B:按照骨阈值定义后成像;C:阈值设置为0时,钛种植体边缘依然成像,验证金属伪影在全阈值值域存在;D:含钛种植体的脱钙灌注标本扫描后成像;E:经血管阻射剂阈值定义成像;F:重建显示伪影形成钛种植体金属管状,并引起植体周围噪点。
图5 小鼠股骨脱钙与未脱钙的钛种植体周围组织扫描分析界面设置 A:脱钙完成的C57BL/6小鼠股骨组织,可见血管造影成像,紫色圈标示定量分析区域,该区域被定义为种植体周围组织的感兴趣区域(VOI);B:未脱钙的C57BL/6小鼠股骨组织,高密度影像为血管或骨组织成像。
图6 不同Gaussian滤镜降噪参数对显微计算机断层扫描(Micro-CT)小鼠血管重建图像的影响 A:σ=1,support=1;B:σ=0.5,support=1。
图7 不同阈值对显微计算机断层扫描(Micro-CT)小鼠血管重建图像的影响 A:阈值设置80;B:阈值设置75。
图8 显微计算机断层扫描(Micro-CT)定量分析灌注实验组小鼠不同时间点的种植体周围血管参数示意图 A:灌注实验组股骨植入钛棒的干骺端血管重建图;B:种植体周围组织(300 μm)血管重建图及彩色管径分布图。
图9 显微计算机断层扫描(Micro-CT)定量分析小鼠灌注实验组术后不同时间点股骨内种植体周围血管参数比较统计图(aP<0.05,bP<0.001)
表1 灌注实验组小鼠各时间点股骨内种植体周围血管相关参数的定量结果比较(±s
图10 不同时间点小鼠种植体周骨组织苏木精-伊红染色及CD31免疫组织化学染色结果 A:种植体周骨组织苏木精-伊红染色;B:种植体周骨组织CD31免疫组化染色(红色虚线标记原种植体边缘)。
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