Objective

To establish a method for investigating the permeability of calcified cartilage zone (CCZ) and to observe solute transport between articular cartilage (AC) and subchondral bone (SB) through intact CCZ in vivo.

Design

We developed a novel fixing device combined with un-decalcified fluorescence observation method to address the permeability of CCZ in live mice. Twenty-four Balb/c female mice aged 1 to 8 months were used to observe the development of CCZ. Eighty-four Balb/c female mice (aged 1 or 6 months) with mature or immature CCZ of distal femur were used to investigate the permeability of intact CCZ in vivo. Diffusivity of rhodamine B (476 Da) and tetramethyl-rhodamine isothicyanate-dextran (TRITC-Dextran, 20 kDa) was tested from AC to SB in 0 minutes, 1 minute, 15 minutes, 30 minutes, 1 hour, and 2 hours. None diffused knee joints (0 minutes) served as blank control, while in vitro immersion of distal femurs in rhodamine B or TRITC-Dextran for 72 hours served as positive control.

Results

CCZ was well developed in 6-month mice. Both tracers penetrated immature CCZ down to SB in less than 1 hour in live mice, while the diffusion of both tracers decreased rapidly at tidemark in all testing time points.

Conclusion

Current study provided direct evidence of blocking effect of CCZ in solute transportation during short diffusion period in live animal, indicating the important role of CCZ in joint development and microenvironment maintenance.

中文翻译：

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活体小鼠关节软骨与软骨下骨之间溶质运输的观察。

 客观的

建立一种研究钙化软骨区（CCZ）渗透性的方法，并观察体内溶质通过完整CCZ在关节软骨（AC）和软骨下骨（SB）之间的转运。

 设计

我们开发了一种新型固定装置结合未脱钙荧光观察方法来解决活体小鼠 CCZ 的通透性问题。采用24只1～8月龄Balb/c雌性小鼠观察CCZ的发育情况。使用84只股骨远端具有成熟或未成熟CCZ的Balb/c雌性小鼠（1月龄或6月龄）来研究完整CCZ的体内通透性。测试了罗丹明 B (476 Da) 和四甲基罗丹明异硫氰酸酯-葡聚糖 (TRITC-Dextran, 20 kDa) 在 0 分钟、1 分钟、15 分钟、30 分钟、1 小时和 2 小时内从 AC 到 SB 的扩散性。未扩散的膝关节(0分钟)作为空白对照，而股骨远端在罗丹明B或TRITC-葡聚糖中体外浸泡72小时作为阳性对照。

 结果

CCZ 在 6 个月大的小鼠中发育良好。在活体小鼠中，两种示踪剂在不到 1 小时的时间内就渗透到未成熟的 CCZ 至 SB，而两种示踪剂的扩散在所有测试时间点的潮汐标记处均迅速下降。

 结论

目前的研究提供了CCZ在活体动物短扩散期内对溶质运输的阻断作用的直接证据，表明CCZ在关节发育和微环境维持中的重要作用。