ETH Zurich, Institute for Biomechanics, Zurich, Switzerland.
University Medical Center Hamburg-Eppendorf, Department of Osteology and Biomechanics, Hamburg, Germany.
J Biomed Opt. 2018 Nov;23(11):1-6. doi: 10.1117/1.JBO.23.11.116504.
Medical knowledge of the skeleton including its structures has improved constantly over the past decades. Advanced imaging methods, mechanical testing and optical techniques have revealed insights into bone architecture and composition. Most of these advancements were possible due to the ex vivo investigation of biological tissues. Investigations of fresh tissue are generally preferred over preserved or fixed samples. However, chemical fixation is sometimes inevitable due to histological procedures or logistical reasons. The aim of this study was to investigate whether short-term chemical fixation with formaldehyde affects bone quality parameters obtained from Raman spectroscopy and if these effects last for intermediate sample storage of several hours. As formaldehyde induces cross-links to the organic components in bone tissue, we hypothesized that collagen-related parameters are particularly affected. Femurs of eight 17-week-old C57BL/6 mice were extracted and divided into two groups (N = 8 / group). Samples of the first group were fixed by immersion in 4% formaldehyde (PFA-solution) for 12 h at 4°C (fixed group) while samples of the second group were left untreated (unfixed group). Raman spectroscopy was performed, and repeated after 4 h, to assess whether intermediate storage time influenced the obtained results. Based on resultant spectra, mineral-to-matrix ratio, carbonate-to-phosphate ratio, carbonate-to-amide I ratio, mineral crystallinity and collagen maturity were determined. Carbonate-to-phosphate ratio was the only parameter showing a significant difference between the first and the subsequent measurements. For both groups, ratios showed a decrease in carbonate substitution compared to the first measurement (percentage decrease: 3.1% in fixed, 4.7% in unfixed). Collagen maturity of samples, which were short-term fixed with formaldehyde, was significantly lower than of fresh, unfixed samples (percentage difference: 3.8%). Our study shows that Raman spectroscopy is able to detect changes in collagen structure initiated by formaldehyde and that changes in short-term fixed samples are minimally influencing bone material properties measured with Raman spectroscopy.
过去几十年中，人们对骨骼的医学知识（包括其结构）的了解不断提高。先进的成像方法、机械测试和光学技术揭示了骨结构和成分的新见解。这些进展大多得益于对生物组织的离体研究。通常，人们更喜欢研究新鲜组织，而不是保存或固定的样本。然而，由于组织学程序或物流原因，有时化学固定是不可避免的。本研究旨在探讨短期福尔马林化学固定是否会影响从拉曼光谱获得的骨质量参数，以及这些影响是否会持续数小时的中间样本储存。由于福尔马林会使骨组织中的有机成分发生交联，我们假设胶原相关参数特别受影响。提取了 8 只 17 周龄 C57BL/6 小鼠的股骨，并将其分为两组（每组 N=8）。第一组的样本用 4%福尔马林（PFA 溶液）在 4°C 下浸泡 12 小时固定（固定组），而第二组的样本则未经处理（未固定组）。进行拉曼光谱分析，并在 4 小时后重复分析，以评估中间储存时间是否会影响获得的结果。根据所得光谱，确定了矿物质与基质的比率、碳酸盐与磷酸盐的比率、碳酸盐与酰胺 I 的比率、矿物质结晶度和胶原成熟度。只有碳酸盐与磷酸盐的比率在第一次和后续测量之间显示出显著差异。对于两组，与第一次测量相比，比率显示出碳酸盐取代的减少（固定组减少 3.1%，未固定组减少 4.7%）。用福尔马林短期固定的样本的胶原成熟度明显低于新鲜、未固定的样本（差异百分比：3.8%）。我们的研究表明，拉曼光谱能够检测到由福尔马林引发的胶原结构变化，并且短期固定样本的变化对用拉曼光谱测量的骨材料性质的影响最小。
