The subchondral bone has no mechanical changes after acute injury for 2 weeks

doi:10.3969/j.issn.2095-4344.2013.20.004

Abstract

Abstract:

BACKGROUND: Previous studies mainly focused on the degenerative cartilage damage and repair mechanisms of the subchondral bone. However, studies on the histopathology and biomechanics of the subchondral bone after acute cartilage injury are few.
OBJECTIVE: To investigate morphology, molecular biology and biomechanical changes of the subchondral bone after acute cartilage injury.
METHODS: Twenty-four healthy adult New Zealand rabbits were used to establish the femoral head cartilage defect model. The specimens of femoral head cartilage and subchondral bone were collected immediately and at 4, 7 and 14 days after modeling, and then the morphological changes of femoral head cartilage and cartilage were generally observed. Safranin-fast green staining was used to observe the morphological changes of femoral head cartilage and subchondral bone; expression of osteoprotegerin/nuclear factor kappa B receptor activating factor ligand was tested with immunohistochemistry; ultrastructural changes and the biomechanical assessment of the subchondral bone were observed with Micro-CT scan and biomechanical assessment, respectively.
RESULTS AND CONCLUSION: Gross visualization revealed that degenerative aggravation of the cartilage defect which was evidenced by enlargement of the area and the depth of the defect at 7 days after modeling. The reduced cartilage thickness and subchondral trabecular bone absorption were observed at 7 days after modeling with the staining methods. Imunohistochemistry and immunofluorescence found that the expression of osteoprotegerin was decreased significantly at 7-14 days after modeling, while the expression of nuclear factor kappa B receptor activating factor ligand was increased significantly, and the ratio of osteoprotegerin/nuclear factor kappa B receptor activating factor ligand was reduced, indicating that bone conversion was weak or even reversed. Micro-CT analysis demonstrated that the trabecular number and spacing of the subchondral bone were greatly reduced, while the number of the trabeculae was increased with reduced permeability at 7-14 days after modeling. Biomechanical test showed that there was no significant difference in the compressive strength and elastic modulus. The results indicate that notable histomorphologic change and down-regulation of turnover of the subchondral bone were identified at 7 days after cartilage injury, which resulted in aggressive damage to the articular cartilage. However, no mechanical changes of subchondral bone observed in 2 weeks after modeling, and the therapeutic strategy based on these findings may present potential targets for cartilage regeneration.

Key words: tissue construction, cartilage tissue construction, articular cartilage, subchondral bone, reconstruction and absorption, trabecular bone, morphology, biomechanics, National Natural Science Foundation of China

CLC Number:

R318

Guo Xin-yu, Zhang Yuan, Wang Zhi-bing, Qin Chuan, Hao Yong, Zhang Yu-mei, Zhang Xia. The subchondral bone has no mechanical changes after acute injury for 2 weeks[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(20): 3635-3642.

Figures/Tables 5

直视状态下对软骨缺损造模后进行观察，可见造模后即刻组膝关节面光滑完整，有光泽、无缺损形成；造模后4，7，14 d组可见膝关节软骨面破溃，完整性遭到破坏，呈现明显颗粒感，表面晦涩，失去光泽。随着取材时间的推移，软骨造模区缺损面积扩大，深度增加，缺损周围软骨灰白暗淡、不透明。利用MIG2000软件测定其缺损面积及深度，发现造模后7-14 d缺损面积和深度比较差异有显著性意义(P < 0.05)。 2.3 显微形态学分析结果采用番红-固绿染色法观察软骨及软骨下骨的组织学变化，由图2可见关节软骨和软骨下骨分别被标记为红色和绿色。镜下观察、采样结合MIG2000软件定量可对关节软骨和软骨下骨进行量化评价，见图2。可见软骨缺损模型后，关节软骨厚度不断下降，至造模后4，7，14 d分别为造模后即刻组的85.2%，63.7%，38.5%(P < 0.05)。而软骨下正常骨小梁结构逐渐破坏，表现为软骨下骨组织填充物比逐渐增加，统计结果显示分别增加0.5，1.1，1.8倍，差异无显著性意义(P > 0.05)。 2.4　骨转换分析结果骨保护素和核因子κB受体活化因子配体分别为成骨-破骨活动标志物，两者在不同时期软骨下骨中的表达，可以作为指标来反映软骨下骨内骨吸收/骨重建的动态变化。实验采用免疫组织化学和免疫荧光法分别评价骨保护素/核因子κB受体活化因子配体，见图3A。紫色着色区为细胞核，棕褐色着色区反映两种物质在细胞间质的表达。利用IPP软件对阳性区域进行量化测定，发现软骨下骨中骨转化标志物的表达发生早期改变。与造模后即刻相比，造模后4，7，14 d组骨保护素/核因子κB受体活化因子配体表达随时间推移逐渐下降，造模后4，7，14 d的表达量分别为造模后即刻的78.1%，43.2%，40%。与之对应，免疫荧光法测定亦证实了成骨功能的削弱和破骨活动的增强，造模后4，7，14 d的骨保护素/核因子κB受体活化因子配体分别下调了48.7%，71.2%，61.7%。 2.5 软骨下骨微观形态学分析结果随着病变时间进展，软骨下骨正常的骨小梁结构逐渐消失，表现为间隙变小，通透性差。采集数据发现：骨小梁间隙及骨小梁厚度减少、骨小梁数目增加，Mean/Density(组织块的填充密度)比值越来越大，骨小梁连接密度显著下降，结构模型指数降低，造模后7-14 d变化最大，差异有显著性意义(P < 0.05)。见图4，表1。"

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