股骨头坏死早期的关节软骨改变
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摘要
目的:探讨早期股骨头坏死关节软骨的改变,为深入了解股骨头坏死的病理机制及制定合理治疗提供实验与临床依据。
材料和方法:本研究分为三部分。(1)建立股骨头软骨下骨缺损动物模型,在不同时间点对实验动物股骨头软骨进行组织学及组织化学分析。18只杂种成年犬一侧股骨头制造骨缺损模型,对侧作为对照。分别于4、12、24周各处死6只动物取材。取材前行股骨头X线检查,大体标本检查。应用HE染色、天狼星红染色及甲苯胺蓝染色方法行关节软骨组织学检查,应用氨基酸分析仪及分光光度计行关节软骨组织化学定量检测并行统计学分析。(2)采用软骨延迟增强核磁成像(delayedgadolinium-enhanced magnetic resonance imaging of cartilage-dGEMRIC)技术,对Ⅱ、Ⅲ期成人股骨头坏死的关节软骨进行检测。20髋激素性股骨头坏死患者中,以国际骨坏死与循环协会(ARCO)分期标准,股骨头坏死Ⅱ期患者为11髋,股骨头坏死Ⅲ期患者为9髋,5名健康志愿者作测试为对照组。对每名受试者进行钆增强延迟核磁共振(1.5T)扫描,分别获得T1Gd数据,应用Matlab和mrimapper软件对每位受试者的影像数据进行分析。(3)软骨生物标记物测定:分别收集10名患者和5名健康志愿者空腹静脉血,保存在-20℃冰箱中,于1000xg离心20分钟,取上清液进行血清软骨寡聚基质蛋白(Cartilage oligomeric matrix protein—COMP),硫酸角质素(Keratan sulfate—KS)检测。
结果:(1)造模术后12周,可见到骨缺损上部邻近软骨细胞减少,细胞核变小,软骨基质染色变浅。术后24周,可见到细胞排列不整齐,呈细胞簇状。紧邻骨缺损开口处软骨浅表层出现裂隙及纤维化改变。潮线模糊、中断,部分切片出现软骨血管形成改变,可见血管突破潮线现象。邻近骨缺损处软骨糖胺聚糖(glycosaminoglycan,GAG)减少。(2)股骨头坏死Ⅱ期患者的MRIT1Gd值(441.13±41.32)与健康志愿者的T1Gd值(544.2±27.83)有显著差别(P＜0.001)。股骨头坏死Ⅲ期患者的T1Gd值(363.11±56.39)与健康志愿者的T1Gd值也有显著差别(P＜0.001)。股骨头坏死Ⅲ期与股骨头坏死Ⅱ期患者的T1Gd值比较下降17.7%,两者有显著性差别(P＜0.05)。(3)患者血清中KS浓度(123.28ng╱ml±17.93,N=10)与健康对照者血清中KS浓度(105.3ng╱ml±14.97,N=5)差异无统计学意义(P＞0.05)及患者血清中软骨寡聚基质蛋白浓度(588.44 ng╱ml±99.68,N=10)与健康对照者血清中COMP浓度(542.5 ng╱ml±28.29,N=5)未见显著差别(P＞0.05),差别无统计学意义。实验组血清中硫酸角质素及软骨寡聚基质蛋白的含量与对照组比较,虽有上升趋势,但差别无统计学意义。
结论:股骨头坏死早期患者的关节软骨已经开始发生代谢变化,表现为关节软骨内的糖胺聚糖(GAG)丢失,说明骨坏死早期可以影响软骨的代谢和功能,并随着坏死程度加重而加大。
Objective:To detect the articular cartilage changes of the hip joint at early stage of the osteonecrosis of femoral head(ONFH),and provide theoretic evidence for understanding the pathomechanism of ONFH and performing rational therapy.
Methods:This study was divided into three parts,(1) The animal model of subchondral bone detect of femoral head was established,articular cartilage changes of femoral head were evaluated by histological and histochemical assay at various time point.18 adult hybrid dogs were applied for bone defect model of femoral head.Of the femoral heads, the left was applied for bone defect model,the right for shame-operation.5,5,and 8 animals were sacreficed respectively at 4,12 and 24 weeks after operation and the femoral heads were received for examinations.The X-rays films were taken just before the animals were sacreficed.After the cartilage specimens were decalcified,dehydrated and embedded,6 u thick sections were made.They were stained with hematoxylin-eosin, sirius-red and toilude blue.The biochemical assay were performed.(2) the articular cartilage changes of stageⅡ、Ⅲof ONFH were determined by delayed gadolinium-enhanced magnetic resonance imaging of cartilage(dGEMRIC).5 asymptomatic volunteers and 20 patients with ONFH participated in the test.According to ARCO classification of ONFH,stageⅡin 11 cases,stageⅢin 9 cases,they were all steroid type.All participants underwent the tests by dGEMRIC(1.5 T),and the data of T1Gd were collected and analyzed by means of Matlab and mrimapper software.(3) The biological markers of cartilage were analyzed to determine the cartilage metabolic changes of ONFH.Serum levels of Cartilage oligomeric matrix protein(COMP) and Keratan sulfate(KS) were quantified with a sandwich-ELISA for ten cases of ONFH and five asymptomatic volunteers respectively.
Results:(1) At 12 and 24 weeks after operation for bone defect,cartilage histology and biochemistry demonstrated the signs of articular cartilage pathological changes.The arrangement of chondrocytes was in disorder.The number of chondrocytes decreased. The cartilage surface lost its smoothness,and these changes were more severe seen in the area next to the bone defect.Fibrosis and vasiformation were found.Tidemark had the changes of discontinuation and veil.Glycosaminoglycans decreased.(2) The values of T1Gd obtained from the patients with stageⅡ,ⅢONFH,and the healthy volunteers were 441.13±41.32,363.11±56.39,and 544.2±27.83 respectively.The values of T1 Gd in the patients of stageⅡ,ⅢONFH were lower than in volunteers,there was a significant difference in T1Gd between the patients of stageⅡorⅢand the healthy volunteers (P＜0.001).The values of T1Gd of the patients of stageⅢwas 17.7%lower than the patients of stageⅡ,and the difference between two stages was significant statistically (P＜0.05).(3) The serum level of KS was 123.28ng/ml±17.93,105.3ng/ml±14.97 in patients and volunteers respectively;and serum level of COMP was 588.44 ng/ml±99.68, 542.5 ng/ml±28.29 in patients and volunteers respectively.Compared with the volunteer group,there was an increase tendency in the serum level of both KS and COMP,but without statistic significant difference.
Conclusion:At early satge of ONFH,articular cartilage of the femoral head appears abnormal metabolic changes,represented by the loss of GAG in the articular cartilage, and aggravated as worsening of ONFH.This demonstrates the metabolism and function of the articular cartilage can be influenced at early stages of ONFH,but the mechanism is not clear and further study is needed.
材料和方法:本研究分为三部分。(1)建立股骨头软骨下骨缺损动物模型,在不同时间点对实验动物股骨头软骨进行组织学及组织化学分析。18只杂种成年犬一侧股骨头制造骨缺损模型,对侧作为对照。分别于4、12、24周各处死6只动物取材。取材前行股骨头X线检查,大体标本检查。应用HE染色、天狼星红染色及甲苯胺蓝染色方法行关节软骨组织学检查,应用氨基酸分析仪及分光光度计行关节软骨组织化学定量检测并行统计学分析。(2)采用软骨延迟增强核磁成像(delayedgadolinium-enhanced magnetic resonance imaging of cartilage-dGEMRIC)技术,对Ⅱ、Ⅲ期成人股骨头坏死的关节软骨进行检测。20髋激素性股骨头坏死患者中,以国际骨坏死与循环协会(ARCO)分期标准,股骨头坏死Ⅱ期患者为11髋,股骨头坏死Ⅲ期患者为9髋,5名健康志愿者作测试为对照组。对每名受试者进行钆增强延迟核磁共振(1.5T)扫描,分别获得T1Gd数据,应用Matlab和mrimapper软件对每位受试者的影像数据进行分析。(3)软骨生物标记物测定:分别收集10名患者和5名健康志愿者空腹静脉血,保存在-20℃冰箱中,于1000xg离心20分钟,取上清液进行血清软骨寡聚基质蛋白(Cartilage oligomeric matrix protein—COMP),硫酸角质素(Keratan sulfate—KS)检测。
结果:(1)造模术后12周,可见到骨缺损上部邻近软骨细胞减少,细胞核变小,软骨基质染色变浅。术后24周,可见到细胞排列不整齐,呈细胞簇状。紧邻骨缺损开口处软骨浅表层出现裂隙及纤维化改变。潮线模糊、中断,部分切片出现软骨血管形成改变,可见血管突破潮线现象。邻近骨缺损处软骨糖胺聚糖(glycosaminoglycan,GAG)减少。(2)股骨头坏死Ⅱ期患者的MRIT1Gd值(441.13±41.32)与健康志愿者的T1Gd值(544.2±27.83)有显著差别(P＜0.001)。股骨头坏死Ⅲ期患者的T1Gd值(363.11±56.39)与健康志愿者的T1Gd值也有显著差别(P＜0.001)。股骨头坏死Ⅲ期与股骨头坏死Ⅱ期患者的T1Gd值比较下降17.7%,两者有显著性差别(P＜0.05)。(3)患者血清中KS浓度(123.28ng╱ml±17.93,N=10)与健康对照者血清中KS浓度(105.3ng╱ml±14.97,N=5)差异无统计学意义(P＞0.05)及患者血清中软骨寡聚基质蛋白浓度(588.44 ng╱ml±99.68,N=10)与健康对照者血清中COMP浓度(542.5 ng╱ml±28.29,N=5)未见显著差别(P＞0.05),差别无统计学意义。实验组血清中硫酸角质素及软骨寡聚基质蛋白的含量与对照组比较,虽有上升趋势,但差别无统计学意义。
结论:股骨头坏死早期患者的关节软骨已经开始发生代谢变化,表现为关节软骨内的糖胺聚糖(GAG)丢失,说明骨坏死早期可以影响软骨的代谢和功能,并随着坏死程度加重而加大。
Objective:To detect the articular cartilage changes of the hip joint at early stage of the osteonecrosis of femoral head(ONFH),and provide theoretic evidence for understanding the pathomechanism of ONFH and performing rational therapy.
Methods:This study was divided into three parts,(1) The animal model of subchondral bone detect of femoral head was established,articular cartilage changes of femoral head were evaluated by histological and histochemical assay at various time point.18 adult hybrid dogs were applied for bone defect model of femoral head.Of the femoral heads, the left was applied for bone defect model,the right for shame-operation.5,5,and 8 animals were sacreficed respectively at 4,12 and 24 weeks after operation and the femoral heads were received for examinations.The X-rays films were taken just before the animals were sacreficed.After the cartilage specimens were decalcified,dehydrated and embedded,6 u thick sections were made.They were stained with hematoxylin-eosin, sirius-red and toilude blue.The biochemical assay were performed.(2) the articular cartilage changes of stageⅡ、Ⅲof ONFH were determined by delayed gadolinium-enhanced magnetic resonance imaging of cartilage(dGEMRIC).5 asymptomatic volunteers and 20 patients with ONFH participated in the test.According to ARCO classification of ONFH,stageⅡin 11 cases,stageⅢin 9 cases,they were all steroid type.All participants underwent the tests by dGEMRIC(1.5 T),and the data of T1Gd were collected and analyzed by means of Matlab and mrimapper software.(3) The biological markers of cartilage were analyzed to determine the cartilage metabolic changes of ONFH.Serum levels of Cartilage oligomeric matrix protein(COMP) and Keratan sulfate(KS) were quantified with a sandwich-ELISA for ten cases of ONFH and five asymptomatic volunteers respectively.
Results:(1) At 12 and 24 weeks after operation for bone defect,cartilage histology and biochemistry demonstrated the signs of articular cartilage pathological changes.The arrangement of chondrocytes was in disorder.The number of chondrocytes decreased. The cartilage surface lost its smoothness,and these changes were more severe seen in the area next to the bone defect.Fibrosis and vasiformation were found.Tidemark had the changes of discontinuation and veil.Glycosaminoglycans decreased.(2) The values of T1Gd obtained from the patients with stageⅡ,ⅢONFH,and the healthy volunteers were 441.13±41.32,363.11±56.39,and 544.2±27.83 respectively.The values of T1 Gd in the patients of stageⅡ,ⅢONFH were lower than in volunteers,there was a significant difference in T1Gd between the patients of stageⅡorⅢand the healthy volunteers (P＜0.001).The values of T1Gd of the patients of stageⅢwas 17.7%lower than the patients of stageⅡ,and the difference between two stages was significant statistically (P＜0.05).(3) The serum level of KS was 123.28ng/ml±17.93,105.3ng/ml±14.97 in patients and volunteers respectively;and serum level of COMP was 588.44 ng/ml±99.68, 542.5 ng/ml±28.29 in patients and volunteers respectively.Compared with the volunteer group,there was an increase tendency in the serum level of both KS and COMP,but without statistic significant difference.
Conclusion:At early satge of ONFH,articular cartilage of the femoral head appears abnormal metabolic changes,represented by the loss of GAG in the articular cartilage, and aggravated as worsening of ONFH.This demonstrates the metabolism and function of the articular cartilage can be influenced at early stages of ONFH,but the mechanism is not clear and further study is needed.
引文
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