HIGH-RESOLUTION 3D IMAGING OF BOVINE TAIL INTERVERTEBRAL DISC DEGENERATION USING IODINE-ENHANCED X-RAY MICROSCOPY

V. Peitso, S. Das Gupta, S. Kauppinen, M. Risteli, M. Finnilä, A. Mobasheri
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Abstract

INTRODUCTION

The vertebral endplates of the intervertebral disc (IVD) consist of two structurally distinct layers: the cartilaginous endplate (CEP) and the bony endplate (BEP). While most research on IVD degeneration has focused on the biochemical or biomechanical failures of the annulus fibrosus (AF) and nucleus pulposus (NP), the physiology and microstructure of the CEP have often been overlooked. To address this gap, we employed iodine-enhanced X-ray microscopy (XRM) in a bovine tail IVD degeneration model. This approach enabled the simultaneous visualization of soft and hard tissues, with a specific focus on the CEP.

OBJECTIVE

1) To simultaneously visualize soft and hard tissues in IVDs, with a specific focus on detecting changes in the CEP using iodine-enhanced XRM. 2) To validate the observed structural changes through histological analysis.

METHODS

34 IVDs with intact vertebral endplates were harvested from six fresh bovine tails. Samples were cultured in Dulbecco’s Modified Eagle Medium (DMEM) for 11 days under unloaded conditions. On day one, approximately 70-100 μL of chondroitinase ABC (chABC, 0.5 U/mL), a pro-inflammatory cytokine cocktail containing interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) (each at 100 ng/mL), or a sham control solution of phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA) was injected into the NP using a 21G needle. Additional control samples received no injection. On day 11, IVDs were fixed in 4% formaldehyde and dehydrated. Samples were immersed in 1% (w/v) iodine (I2) in 100% ethanol and stained for a minimum of two weeks. Following staining, samples were washed, embedded in 1% agarose, and imaged with an XRM (Zeiss Xradia Versa 610; source voltage: 60kV; exposure: 4-6 sec; voxel size: 9.9-15.6 µm). Post-imaging, iodine was removed, and samples were decalcified and paraffin-embedded. Thin sections (7-10 µm) were prepared and stained with hematoxylin and eosin (H&E) and safranin-O and fast green. Reconstituted XRM image stacks were processed using built-in noise filtering software (Zeiss). Dragonfly 3D world (Comet) software was used for visualization and segmentation. XRM images were qualitatively compared with histological sections to assess changes in soft and hard tissues (Figures 1 and 2).

RESULTS

The interface between mineralized and non-mineralized cartilage (tidemark) was visualized using XRM, enabling the identification of calcified cartilage and CEP (Figure 1). Iodine-based contrast provided sufficient resolution to detect structural malalignments among the BEP, CEP, and NP (Figure 2). Notably, even sham injections with PBS induced degenerative changes in the disc.

CONCLUSION

Non-destructive iodine-enhanced XRM enables clear visualization of the CEP, providing sufficient contrast to simultaneously assess structural changes in both soft and hard tissues. This approach offers a powerful tool for evaluating IVD degeneration on ex vivo models.
利用碘增强x射线显微镜对牛尾椎间盘退变进行高分辨率三维成像
椎间盘终板(IVD)由两层结构不同的椎体终板组成:软骨终板(CEP)和骨终板(BEP)。虽然大多数关于IVD退变的研究都集中在纤维环(AF)和髓核(NP)的生化或生物力学失效上,但CEP的生理和微观结构往往被忽视。为了解决这一差距,我们在牛尾IVD变性模型中使用了碘增强x射线显微镜(XRM)。该方法能够同时显示软硬组织,并特别关注CEP。目的1)同时显示ivd中的软硬组织,特别关注使用碘增强XRM检测CEP的变化。2)通过组织学分析验证观察到的结构变化。方法从6只新鲜牛尾中取出34只具有完整椎终板的ivd。样品在Dulbecco 's Modified Eagle Medium (DMEM)中脱模培养11天。第一天,用21G针将约70-100 μL的软骨素酶ABC (chABC, 0.5 U/mL)、含有白细胞介素-1β (IL-1β)和肿瘤坏死因子α (TNF-α)的促炎细胞因子鸡尾酒(各100 ng/mL)或含有0.1%牛血清白蛋白(BSA)的磷酸盐缓冲盐水(PBS)假对照溶液注射到NP中。另外的对照样本没有注射。第11天,将ivd固定在4%甲醛中并脱水。样品浸泡在1% (w/v)碘(I2)和100%乙醇中,染色至少两周。染色后,清洗样品,包埋在1%琼脂糖中,用XRM成像(蔡司Xradia Versa 610;源电压:60kV;曝光:4-6秒;体素尺寸:9.9-15.6µm)。成像后,去除碘,样品脱钙并包埋石蜡。制备7-10µm薄片,用苏木精和伊红(H&;E)、藏红花素- o和快绿染色。重建的XRM图像堆栈使用内置的噪声滤波软件(蔡司)进行处理。使用Dragonfly 3D world (Comet)软件进行可视化和分割。将XRM图像与组织学切片进行定性比较,评估软硬组织的变化(图1和2)。结果XRM显示了矿化软骨和非矿化软骨之间的界面(潮汐标记),可以识别钙化软骨和CEP(图1)。基于碘的对比提供了足够的分辨率来检测BEP、CEP和NP之间的结构失调(图2)。值得注意的是,即使是假注射PBS也会引起椎间盘的退行性改变。结论非破坏性碘增强XRM能够清晰地显示CEP,提供足够的对比,同时评估软硬组织的结构变化。这种方法为评估体外模型的IVD退化提供了强有力的工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Osteoarthritis imaging
Osteoarthritis imaging Radiology and Imaging
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