Development of a Mouse Model of Ischemic Osteonecrosis
Development of a Mouse Model of Ischemic Osteonecrosis
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DOI：
10.1007/s11999-015-4172-6
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发表时间：
2015-04-01
影响因子：
4.2
通讯作者：
Kim, Harry K. W.
中科院分区：
文献类型：
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作者：
Kamiya, Nobuhiro;Yamaguchi, Ryosuke;Kim, Harry K. W.
可靠的缺血性骨坏死小鼠模型的可用性可以加速缺血性骨坏死后刺激骨愈合的新治疗策略的开发;然而，目前还没有缺血性骨坏死的小鼠模型。为了开发缺血性骨坏死的外科小鼠模型，我们提出了以下问题：（1）如果烧灼有助于远端股骨骨骺血液供应的血管，本研究通过显微血管造影技术观察了小鼠股骨头远端骨骺的供血血管，并采用显微外科技术烧灼了4条血管，以诱导缺血性骨坏死。使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）和计数三个连续切片中的空陷窝，对骨小梁中的细胞死亡进行组织学评估。使用图像分析软件进行破骨细胞和成骨细胞数量的定量。使用micro-CT对股骨远端骨骺的畸形和松质骨参数进行形态学评估。我们确定了膝关节周围的四条血管，这些血管必须被烧灼以诱导股骨远端骨骺的完全缺血性骨坏死。骨骺组织学切片的定性评估显示，1周时骨髓细胞核染色丧失、骨髓结构紊乱和血管坏死。到2周时，观察到坏死骨髓空间的血管组织浸润，在4周和6周时，坏死骨髓空间的血管组织浸润逐渐增加。TUNEL染色显示诱导缺血后24小时骨髓和松质骨中广泛的细胞死亡。诱导缺血后1周，小梁骨中TUNEL阳性骨细胞的平均百分比从对照组的2%+/-A1%增加到缺血组的98%+/-A3%的峰值（平均差异，96%; 95%CI，81%-111%; p <0.0001）。空骨陷窝的平均百分比从对照组的1%+/-A1%增加到缺血组在4周时的峰值78%+/-A15%（平均差异，77%; 95%CI，56%-97%; p <0.0001）。定量分析显示，与对照组相比，缺血组在1、2和4周时每个骨表面的破骨细胞平均数量减少（分别为p <0.0001、p <0.0001和p = 0.02）。破骨细胞的平均数量在6周时增加至与对照组相似的水平（p = 0.23）。与对照组相比，缺血组在1、2和4周时每个骨表面的成骨细胞数量减少（各p <0.0001）。6周时，成骨细胞的平均数量也增加至与对照组相似的水平（p = 0.91）。从2到6周，缺血组通过micro-CT评估的平均骨体积百分比低于对照组。在第2、4和6周，对照组和缺血组之间的骨体积百分比的平均差异为5.5%。（95% CI，0.9%-10.2%; p = 0.01）、5.3%（95% CI，0.6%-9.9%; p = 0.02）和6.0%（95% CI，1.1%-10.9%; p = 0.008）。6周时观察到股骨远端骨骺畸形，对照组的平均骨骺高宽比为0.74 +/-A0.03，而缺血组为0.66 +/-A0.06（平均差，0.08; 95%CI，0.00 - 0.16; p = 0.03）我们开发了一种新的缺血性骨坏死小鼠模型，该模型在股骨远端骨骺中产生广泛的细胞死亡，该骨骺随着时间的推移发生畸形。新的小鼠模型可能是一个有用的工具，以测试潜在的治疗策略，以改善缺血性骨坏死后的骨愈合。
Availability of a reliable mouse model of ischemic osteonecrosis could accelerate the development of novel therapeutic strategies to stimulate bone healing after ischemic osteonecrosis; however, no mouse model of ischemic osteonecrosis is currently available.To develop a surgical mouse model of ischemic osteonecrosis, we asked, (1) if the blood vessels that contribute to the blood supply of the distal femoral epiphysis are cauterized, can we generate an osteonecrosis mouse model; (2) what are the histologic changes observed in this mouse model, and (3) what are the morphologic changes in the model.We performed microangiography to identify blood vessels supplying the distal femoral epiphysis in mice, and four vessels were cauterized using microsurgical techniques to induce ischemic osteonecrosis. Histologic assessment of cell death in the trabecular bone was performed using terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) and counting the empty lacunae in three serial sections. Quantitation of osteoclast and osteoblast numbers was performed using image analysis software. Morphologic assessments of the distal femoral epiphysis for deformity and for trabecular bone parameters were performed using micro-CT.We identified four blood vessels about the knee that had to be cauterized to induce total ischemic osteonecrosis of the distal femoral epiphysis. Qualitative assessment of histologic sections of the epiphysis showed a loss of nuclear staining of marrow cells, disorganized marrow structure, and necrotic blood vessels at 1 week. By 2 weeks, vascular tissue invasion of the necrotic marrow space was observed with a progressive increase in infiltration of the necrotic marrow space with the vascular tissue at 4 and 6 weeks. TUNEL staining showed extensive cell death in the marrow and trabecular bone 24 hours after the induction of ischemia. The mean percent of TUNEL-positive osteocytes in the trabecular bone increased from 2% +/- A 1% in the control group to a peak of 98% +/- A 3% in the ischemic group 1 week after induction of ischemia (mean difference, 96%; 95% CI, 81%-111%; p < 0.0001). The mean percent of empty lacunae increased from 1% +/- A 1% in the control group to a peak of 78% +/- A 15% in the ischemic group at 4 weeks (mean difference, 77%; 95% CI, 56%-97%; p < 0.0001). Quantitative analysis showed that the mean number of osteoclasts per bone surface was decreased in the ischemic group at 1, 2, and 4 weeks (p < 0.0001, < 0.0001, and p = 0.02, respectively) compared with the control group. The mean number of osteoclasts increased to a level similar to that of the control group at 6 weeks (p = 0.23). The numbers of osteoblasts per bone surface were decreased in the ischemic group at 1, 2 and 4 weeks (p < 0.0001 for each) compared with the numbers in the control group. The mean number of osteoblasts also increased to a level similar to that of the control group at 6 weeks (p = 0.91). Mean bone volume percent assessed by micro-CT was lower in the ischemic group compared with the control group from 2 to 6 weeks. The mean differences in the percent bone volume between the control and ischemic groups at 2, 4, and 6 weeks were 5.5% (95% CI, 0.9%-10.2%; p = 0.01), 5.3% (95% CI, 0.6%-9.9%; p = 0.02), and 6.0% (95% CI, 1.1%-10.9%; p = 0.008), respectively. A deformity of the distal femoral epiphysis was observed at 6 weeks with the mean epiphyseal height to width ratio of 0.74 +/- A 0.03 in the control group compared with 0.66 +/- A 0.06 in the ischemic group (mean difference, 0.08; 95% CI, 0.00-0.16; p = 0.03).We developed a novel mouse model of ischemic osteonecrosis that produced extensive cell death in the distal femoral epiphysis which developed a deformity with time.The new mouse model may be a useful tool to test potential therapeutic strategies to improve bone healing after ischemic osteonecrosis.