EX VIVO IMAGING OF DIFFERENT CALCIFICATION TYPES IN POSTERIOR HORN OF HUMAN MENISCUS USING MICRO-COMPUTED TOMOGRAPHY

V.P. Karjalainen , I. Hellberg , A. Turkiewicz , B. Shakya , N. Khoshimova , E. Nevanranta , K. Elkhouly , S. Das Gupta , A. Sjögren , M.A.J. Finnilä , P. Önnerfjord , V. Hughes , J. Tjörnstrand , M. Englund , S. Saarakkala
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引用次数: 0

Abstract

INTRODUCTION

Meniscal calcifications are known to be associated with OA. Specifically, two types of calcifications have been commonly identified in osteoarthritic knees: basic calcium phosphate (BCP) and calcium pyrophosphate (CPP). However, their pathological significance remains largely unclear. Characterizing differences between the calcification types and their deposition patterns inside the meniscus could help in their identification with in vivo imaging modalities and provide a better understanding of the role of meniscal calcifications in the OA disease process.

OBJECTIVE

1) Identify the two different types of calcifications in human meniscus ex vivo in 3D using µCT; 2) Describe the different deposition patterns observed in BCP and CPP calcifications.

METHODS

From the MENIX biobank in Lund, Sweden, we collected 82 posterior horns of medial and lateral menisci from 20 total knee replacement (TKR) patients and 21 deceased donors (50/50% female/male, average age 71 years) for the study. A 5-mm-thick subsection was dissected from the posterior horn, fixed in formalin, dehydrated, and treated with hexamethyldisilazane (HMDS) before air-drying at room temperature overnight. Subsequently, the HMDS-treaded section was imaged with a desktop µCT imaging (SkyScan 1272, Bruker, micro-CT) with the following settings: 60 kV, 166 µA, 2.0 µm voxel size, 3500 ms exposure time, random movement 25 voxels, and without an additional filter. Two different image reconstruction settings were used to maximize the image quality of meniscal soft tissue and calcifications. Pieces of meniscus adjacent to the µCT underwent histological processing and Alizarin Red staining. Calcification types from the histological sections were identified using Raman micro-spectroscopy.

RESULTS

We successfully imaged both meniscal calcification types together with soft tissue in 3D using high-resolution µCT (Figure 1). Based on Raman spectral analysis, out of the 82 menisci, 39 had at least one calcification: 28 had BCP calcifications, 8 had CPP calcifications, and 3 had both. In µCT, BCP calcifications were quantitatively denser, morphologically sharper, more punctuated, smaller in size as well as number, and more spherical than CPPs. Unlike CPPs, BCPs were mainly deposited in the periphery of meniscal tissue, inside complex 3D tears or fibrillations. In contrast, the CPP calcifications formed long rod-like structures, mainly inside the meniscal tissue.

CONCLUSION

Based on the 3D µCT images, BCP calcifications were not found inside the meniscal tissue but in the peripheral area. This could suggest that larger clusters of BCP calcifications found in the meniscus come from the synovial fluid and possibly originate from articular cartilage or bone. Meanwhile, the likely place for CPPs to accumulate and expand within the meniscal tissue is in the fluid channels that follow the circumferential collagen fiber bundles, where they fill the cavity of the channel to form rod-like morphology and have a continuous supply of calcium and other constituents. Additionally, vascular walls were observed to accumulate calcifications, supported by hollow rod-shaped structures that do not follow the circumferential fibers. Potentially, after tearing and degeneration of the meniscus, CPPs may start to accumulate on the surfaces and tears of the meniscus in an amorphous pattern. This qualitative 3D comparison of meniscal calcification patterns may help distinguish them with imaging modalities more easily in the future, as well as provide a better understanding of their role in OA.
人半月板后角不同钙化类型的体外显微计算机断层成像
半月板钙化已知与OA有关。具体来说,两种类型的钙化已被普遍认定为骨关节炎膝关节:碱性磷酸钙(BCP)和焦磷酸钙(CPP)。然而,它们的病理意义在很大程度上仍不清楚。表征半月板内钙化类型及其沉积模式的差异有助于半月板钙化与体内成像模式的识别,并更好地了解半月板钙化在OA疾病过程中的作用。目的1)利用微CT识别人半月板离体三维钙化的两种不同类型;2)描述BCP和CPP钙化的不同沉积模式。方法:研究人员从瑞典隆德MENIX生物银行收集了20例全膝关节置换术(TKR)患者和21例已故供体(男女各占50/50%,平均年龄71岁)的82个内侧和外侧半月板后角。从后角上解剖一个5mm厚的分段,用福尔马林固定,脱水,用六甲基二氮杂烷(HMDS)处理,然后在室温下风干过夜。随后,使用桌面微CT成像(SkyScan 1272, Bruker, micro-CT)对hmds处理的切片进行成像,设置如下:60 kV, 166µa, 2.0µm体素大小,3500 ms曝光时间,随机移动25体素,没有额外的过滤器。采用两种不同的图像重建设置,以最大限度地提高半月板软组织和钙化的图像质量。微CT旁半月板切片进行组织学处理和茜素红染色。组织切片的钙化类型用拉曼显微光谱鉴定。我们使用高分辨率微CT成功地对两种半月板钙化类型以及软组织进行了三维成像(图1)。根据拉曼光谱分析,在82例半月板中,39例至少有一种钙化,28例有BCP钙化,8例有CPP钙化,3例两者都有。在µCT上,BCP钙化密度更大,形态更清晰,标点更多,尺寸和数量更小,比CPPs更球形。与CPPs不同,bcp主要沉积在半月板组织的周围、复杂的3D撕裂或纤颤内。相反,CPP钙化形成长棒状结构,主要在半月板组织内。结论三维微CT图像显示,半月板组织内未见BCP钙化,周围可见BCP钙化。这可能提示半月板内较大的BCP钙化团来自滑液,也可能来自关节软骨或骨。同时,CPPs在半月板组织内可能积聚和扩张的位置是在沿周向胶原纤维束的流体通道中,在那里它们填充通道的腔体形成棒状形态,并持续供应钙和其他成分。此外,血管壁被观察到积累钙化,由不跟随周围纤维的空心杆状结构支撑。在半月板撕裂和退化后,CPPs可能开始以无定形模式积聚在半月板表面和撕裂处。这种半月板钙化模式的定性三维比较可能有助于在未来更容易地与成像方式区分它们,以及更好地了解它们在OA中的作用。
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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