Superficial zone cellularity is deficient in mice lacking lubricin: a stereoscopic analysis.
Superficial zone cellularity is deficient in mice lacking lubricin: a stereoscopic analysis.
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DOI：
10.1186/s13075-016-0967-4
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发表时间：
2016-03-14
影响因子：
4.9
通讯作者：
Jay GD
中科院分区：
文献类型：
--
作者：
Karamchedu NP;Tofte JN;Waller KA;Zhang LX;Patel TK;Jay GD
润滑素是一种由滑膜细胞和软骨细胞分泌的粘液糖蛋白，在降低哺乳动物关节的摩擦系数方面发挥着重要作用。软骨表面摩擦力增加被认为会导致软骨细胞损失；然而，其量化和方法学途径尚未报道。我们采用体视学方法并结合活细胞染色来评估润滑素缺乏的小鼠软骨浅表和上中间区域的细胞损失。使用荧光素二乙酸酯 (FDA)、碘化丙啶 (PI) 和 Hoechst 染色以及共聚焦显微镜对来自润滑素野生型 (Prg4 +/+)、杂合子 (Prg4 +/-) 和敲除 (Prg4 -/-) 小鼠的完整膝盖的股骨髁软骨进行成像。使用 Matlab 分析深度为 14 μm 的共焦图像的三维重建，以确定股骨内侧髁和外侧髁软骨中软骨细胞所占据的体积分数。活软骨细胞体积分数定义为FDA染色的软骨细胞体积/浅表++上中间区的总体积。活软骨细胞和死软骨细胞（总）体积分数定义为FDA + PI染色的软骨细胞体积/浅表 + 上部中间区的总体积。 MicroCT 提供了软骨厚度的正交测量。对激活的 caspase-3 进行免疫组织学分析并进行 TUNEL 染色，以评估 Prg4 突变小鼠中凋亡软骨细胞的存在。与 Prg4 +/+ (p = 0.002) 和 Prg4 +/- (p = 0.002) 同窝小鼠相比，Prg4 -/- 小鼠内侧股骨髁的活软骨细胞体积分数显着较低。 Prg4 +/+ 和 Prg4 +/- 小鼠的内侧髁软骨细胞体积分数没有显着差异（p = 0.82）。外髁软骨细胞体积分数没有观察到显着差异（p > 0.26）。与 Prg4 +/+ (p = 0.02) 和 Prg4 +/- (p = 0.03) 同窝小鼠相比，Prg4 -/- 小鼠的内侧髁软骨厚度增加，与 Prg4 +/+ (p < 0.0001) 和 Prg4 +/- 相比，Prg4 -/- 小鼠的外侧髁突厚度增加(p < 0.0001) 同窝出生，表明多维增加 软骨体积并没有人为地降低内侧髁中的软骨细胞体积分数。与 Prg4 +/+ (p = 0.01) 和 Prg4 +/- (p = 0.04) 同窝小鼠相比，Prg4 -/- 小鼠内侧股骨髁浅层和上中间区软骨中观察到 caspase-3 阳性细胞数量显着增加，而与 Prg4 +/+ 相比，Prg4 -/- 小鼠股骨外侧髁中观察到的 caspase-3 阳性细胞数量显着增加。 (p = 0.02) 和 Prg4 +/- (p = 0.02) 同窝仔鼠。两个髁突中不同 Prg4 基因型的 TUNEL 染色没有显着差异（所有比较中 p > 0.05）。与 Prg4 充足的同窝小鼠相比，Prg4 缺陷小鼠的 Caspase-3 激活增加。 Prg4 的缺失会导致小鼠软骨表面和上中间区软骨细胞的损失，这种损失可以通过一种新颖的图像处理技术进行量化。本文的在线版本 (doi:10.1186/s13075-016-0967-4) 包含补充材料，可供授权用户使用。
Lubricin, a mucinous glycoprotein secreted by synoviocytes and chondrocytes plays an important role in reducing the coefficient of friction in mammalian joints. Elevated cartilage surface friction is thought to cause chondrocyte loss; however, its quantification and methodological approaches have not been reported. We adapted a stereological method and incorporated vital cell staining to assess cellular loss in superficial and upper intermediate zones in lubricin deficient mouse cartilage. The femoral condyle cartilage of the intact knees from lubricin wild type (Prg4 +/+), heterozygote (Prg4 +/-), and knockout (Prg4 -/-) mice was imaged using fluorescein diacetate (FDA), propidium iodide (PI), and Hoechst staining, and confocal microscopy. Three dimensional reconstructions of confocal images to a depth of 14 μm were analyzed using Matlab to determine the volume fraction occupied by chondrocytes in cartilage of both medial and lateral femoral condyles. Living chondrocyte volume fraction was defined as FDA stained chondrocyte volume/total volume of superficial + upper intermediate zone. Living and dead (total) chondrocyte volume fraction was defined as FDA + PI stained chondrocyte volume/total volume of superficial + upper intermediate zone. MicroCT provided an orthogonal measure of cartilage thickness. Immunohistology for activated caspase-3 and TUNEL staining were performed to evaluate the presence of apoptotic chondrocytes in Prg4 mutant mice. Living chondrocyte volume fraction of the medial femoral condyle was significantly lower in Prg4 -/- mice compared to Prg4 +/+ (p = 0.002) and Prg4 +/- (p = 0.002) littermates. There was no significant difference in medial condyle chondrocyte volume fraction between Prg4 +/+ and Prg4 +/- mice (p = 0.82). No significant differences were observed for the chondrocyte volume fraction for the lateral condyle (p > 0.26). Cartilage thickness increased in the medial condyle for Prg4 -/- mice compared to Prg4 +/+ (p = 0.02) and Prg4 +/- (p = 0.03) littermates, and the lateral condyle for Prg4 -/- mice compared to Prg4 +/+ (p < 0.0001) and Prg4 +/- (p < 0.0001) littermates, indicating that a multi-dimensional increase in cartilage volume did not artifactually lower the chondrocyte volume fraction in the medial condyle. Significantly higher number of caspase-3 positive cells were observed in the superficial and upper intermediate zone cartilage of the medial femoral condyle of Prg4 -/- mice compared to Prg4 +/+ (p = 0.01) and Prg4 +/- (p = 0.04) littermates, and the lateral femoral condyle of Prg4 -/- mice compared to Prg4 +/+ (p = 0.02) and Prg4 +/- (p = 0.02) littermates. There were no significant differences in TUNEL staining among different Prg4 genotypes in both condyles (p > 0.05 for all comparisons). Increased Caspase-3 activation is observed in Prg4 deficient mice compared to Prg4 sufficient littermates. Absence of Prg4 induces loss of chondrocytes in the superficial and upper intermediate zone of mouse cartilage that is quantifiable by a novel image processing technique. The online version of this article (doi:10.1186/s13075-016-0967-4) contains supplementary material, which is available to authorized users.