大鼠膝关节负重与非负重区软骨组织学对比分析

Histological compare of articular cartilage of the loading and unloading area in rat knee joints

目的观察大鼠膝关节软骨负重区与非负重区组织形态、基质中蛋白多糖成分和Ⅱ型胶原分布差异。方法 3月龄SD(Sprague-Dawley)大鼠8只,切取双膝关节,沿矢状面整体切片,采用HE染色、p H值1.0和2.5阿尔新蓝染色、番红O染色、阿尔新蓝-番红O复染观察软骨形态结构和基质蛋白多糖成分,并测量软骨厚度,免疫组化检测Ⅱ型胶原分布。分别观察负重区与非负重区软骨形态和基质染色差异,并利用图像分析系统,对基质成分染色深浅进行光密度定量,采用独立样本t检验进行统计分析。结果负重区与非负重区软骨厚度、细胞分布、形态结构均有较大差异。负重区软骨较非负重区明显增厚[胫骨平台(265±39)μm vs.(179±27)μm,t=5.128,P=0.000 2;股骨髁(219±33)μm vs.(132±21)μm,t=6.291,P<0.000 1],且软骨各层结构特征较非负重区更加明显。阿尔新蓝染色结果显示,负重区的非钙化层比非负重区染色更浅(胫骨平台0.073±0.013vs.0.354±0.034,t=21.83,P<0.000 1;股骨髁0.058±0.020 vs.0.131±0.022,t=6.945,P<0.000 1),而番红O深染(胫骨平台0.246±0.013 vs.0.158±0.034,t=6.838,P<0.000 1;股骨髁0.171±0.020vs.0.086±0.022,t=8.086,P<0.001),差异均有统计学意义,但上述两个区域钙化层的阿尔新蓝染色和番红O染色结果并无统计学差异。阿尔新蓝-番红O复合染色结果显示,负重区软骨的阿尔新蓝染色面积百分比明显少于非负重区[胫骨平台(19.3±5.1)%vs.(88.2±4.2)%,t=29.50,P<0.000 1;股骨髁(36.2±5.8)%vs.(69.5±7.1)%,t=10.27,P<0.000 1],而番红O染色面积百分比却大于非负重区[胫骨平台(72.6±8.2)%vs.(31.2±5.2)%,t=12.06,P<0.000 1;股骨髁(72.1±7.6)%vs.(26.1±8.1)%,t=11.71,P<0.000 1],差异均有统计学意义。负重区软骨非钙化层p H 2.5阿尔新蓝染色明显较p H 1.0阿尔新蓝染色加深。免疫组化未发现负重区与非负重区Ⅱ型胶原平均光密度存在统计学差异(胫骨平台0.033±0.005 vs.0.029±0.006,t=1.449,P=0.169 5;股骨髁0.031±0.009 vs.0.027±0.002,t=1.227,P=0.240 0)。结论胫骨和股骨髁负重区较非负重区厚度增加,软骨各层结构特征更加明显。负重区与非负重区比较,基质中糖胺多糖的含量明显增加,软骨透明质酸含量明显减少,硫酸软骨素和硫酸角质素含量明显增加,而Ⅱ型胶原含量并无差异。说明不同的受力环境可以造成软骨形态结构和基质成分差异,负重区软骨的形态结构更加适应于承受关节的力学作用。同时提示临床用来修复负重区软骨缺损的非负重区软骨缺乏适应负重区力学环境的组织结构,可能是骨软骨移植手术后移植物退化的重要因素。

Objective To observe and analysis the morphology and matrix content in articular cartilage of the loading and unloading area in rat knee joints.Methods Knees of 8 SD (Sprague-Dawley) rats were studied by whole-mount section technique. The sections were stained with haematoxylin-eosin, alcian blue and safranin O to elucidate the morphological difference of loading and unloading area. The cartilage thickness and stain difference were compared. The distribution of collagen typeⅡ was detected by immunohistochemistry. As well, the differences of cartilage morphology and matrix staining between the loading and unloading area were observed, and the optical density was measured by the image analysis system. At last, thet-test method was used for statistical analysis.Results There was great different in the loading and unloading area in the chondrocyte array and cartilage thickness. The cartilage of loading area was thicker than unloading area, with average cartilage thickness (tibia (265±39)μmvs. (179±27)μm, t=5.128,P=0.0002; femur (219±33)μmvs. (132±21)μm,t=6.291,P<0.0001)And the structural characteristics of each layer were more obvious than that of unloading area. Alcian blue staining showed that the non calcified layer of loading area had lighter blue than unloading area, with mean IOD (tibia 0.073±0.013vs. 0.354±0.034,t=21.83,P<0.0001; femur 0.058±0.020vs. 0.131±0.022,t=6.945,P<0.0001), and had deeper safranin O dye than unloading area, with mean IOD (tibia 0.246±0.013vs. 0.158±0.034, t=6.838,P<0.0001; femur 0.171±0.020vs. 0.086±0.022,t=8.086,P<0.001). While, there was no statistical significance in the unloading area. When it comes to Alcian blue and safranin O composite staining, it showed that the cartilage's alcian blue staining percentage of loading area was less than unloading area, with mean IOD [tibia (19.3±5.1)%vs. (88.2±4.2)%,t=29.50,P<0.0001; femur (36.2±5.8)%vs. (69.5±7.1)%,t=10.27,P<0.0001], and an opposite result of the percentage of safranin O staining percentage, with mean IOD (tibia (72.6±8.2)%vs. (31.2±5.2)%,t=12.06,P<0.0001; femur (72.1±7.6)%vs. (26.1±8.1)%,t=11.71,P<0.0001)The alcian blue staining on cartilage calcified layer of loading area was getting deeper when the pH of alcian blue changed from 1.0 to 2.5. There was no significant difference in the average optical density of collagenⅡ between the loading area and unloading area, with mean IOD (tibia 0.033±0.005vs. 0.029±0.006,t=1.449,P=0.1695; femur 0.031±0.009vs. 0.027±0.002,t=1.227, P=0.2400), respectively.Conclusions The loading area of the tibia and the femoral condyle was thicker than that of the unloading area, and a the structural characteristics of different cartilage layers were more obvious as well. Compared with unloading area, cartilage of loading area had a significantly increased content of matrix glycosaminoglycan, chondroitin sulfate and keratan sulfate, and a significantly decreased cartilage hyaluronic acid content, but no difference in the content of collagen typeⅡ. The results show that the different environment in keen joint can cause the difference of cartilage morphology and matrix composition. And the morphology of the cartilage is more suitable for the mechanics of the joint. So that, it is suggested that the unloading area cartilage graft which was used in mosaic plasty to repair the cartilage defects in the loading area was lack of the mechanical structure to adapt the loading biomechanical environment. It may be an important factor in the deterioration of graft after mosaic plasty.