Do formalin fixation and freeze-thaw affect near-infrared Raman spectroscopy of cartilaginous tissue? - An ex vivo analysis of native human articular cartilage
Do formalin fixation and freeze-thaw affect near-infrared Raman spectroscopy of cartilaginous tissue? - An ex vivo analysis of native human articular cartilage
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福尔马林固定和冻融是否影响软骨组织的近红外拉曼光谱？
DOI：
10.1002/jrs.4748
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发表时间：
2015
影响因子：
2.5
通讯作者：
Giuseppe Pezzotti
中科院分区：
文献类型：
--
作者：
Yasuhito Takahashi;Takaaki Shishido;Kengo Yamamoto;Yasunobu Sawaji;Jun NIshida;Giuseppe Pezzotti
近红外(NIR)拉曼微探针谱已被应用于关节软骨细胞外基质生化结构的无创表征，这是软骨病活体诊断应用的又一进步。在大多数研究中，为了在光谱测量之前稳定组织和细胞成分，已经应用了手持活体标本、福尔马林固定或冻融处理。然而，这些预处理操作可能会显著影响软骨光谱的某些目标波段，从而在表征中引入偏差，并可能导致数据误解。在这项研究中，我们评估了福尔马林固定和冻融过程对人股骨软骨拉曼光谱的影响。健康的软骨标本被固定/保存在10%中性缓冲福尔马林溶液中或在−80 °C的冷冻箱中。本研究结果表明，由于胶原和糖胺多聚糖大分子中同时存在福尔马林吸收和水分脱水，福尔马林固定显著影响了软骨标本的近红外拉曼光谱。冻融标本的酰胺I结构中也证实了水的脱水，但程度要小得多。此外，将组织浸泡在磷酸盐缓冲盐水中可最大限度地减少存储诱导的拉曼伪影，但其浸泡在福尔马林固定的标本中的效果有限，主要是由于福尔马林液的信号重叠(即在1046和1492 cm−1处发射)。因此，为了利用近红外拉曼光谱对细胞外基质进行高精度的生化评价，冻融处理比福尔马林固定更适合于人软骨的活体标本。版权所有©2015 John Wiley&Sons，Ltd.
Near‐infrared (NIR) Raman microprobe spectroscopy has been applied to the non‐invasive characterization of the biochemical structure of extracellular matrix in articular cartilage, a step forward along the path ofin vivodiagnostic application of chondropathy. In most studies handlingex vivocartilage specimens, formalin fixation or freeze‐thaw treatments have been applied in order to stabilize tissue and cell constituents prior to spectroscopic measurements. However, these pre‐processing manipulations might significantly affect certain target bands of the cartilage spectra, thus introducing biases in the characterizations, and potentially leading to data misinterpretation. In this study, we evaluated how formalin fixing and freeze‐thaw processes affect Raman spectra from human femur cartilage. Healthy cartilage specimens were fixed/stored either in a 10% neutral buffered formalin solution or in a deep freezer set at −80 °C. The results of this study show that formalin fixation significantly affects the NIR Raman spectra of cartilage specimens due to concurrent formalin absorption and water dehydration within both collagen and glycosaminoglycan macromolecules. Water dehydration was also confirmed in the amide I structure in the frozen‐thawed specimen, but to a much lesser extent. Furthermore, soaking the tissues in phosphate‐buffered saline solution minimized the storage‐induced Raman artifacts, but its immersion had limited effectiveness in formalin‐fixed specimens, predominantly due to an overlap of signals from the formalin liquid (i.e. emitting at 1046 and 1492 cm−1). Therefore, to provide a highly accurate biochemical evaluation of extracellular matrix using NIR Raman spectroscopy, freeze‐thaw processes are more suitable forex vivosamples of human cartilage than formalin fixation. Copyright © 2015 John Wiley & Sons, Ltd.