Chow Dick H, Zheng Lizhen, Tian Li, Ho Kam-Sing, Qin Ling, Guo Xia
Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong Special Administrative Region.
Innovative Orthopaedic Biomaterial and Drug Translational Research Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region.
J Orthop Translat. 2018 Sep 5;17:112-120. doi: 10.1016/j.jot.2018.08.001. eCollection 2019 Apr.
BACKGROUND/OBJECTIVES: Decalcification of bone specimens is necessary for routine paraffin embedding and sectioning. Ethylenediaminetetraacetic acid (EDTA), a chelating agent for decalcification, maintains bone tissue integrity and histological features but requires long decalcification period, especially for cortical bone with dense mineral matrix. We hypothesised that the application of a newly commercially available ultrasound (US) decalcifier would accelerate decalcification of thick cortical bone specimen in EDTA efficiently and that the working temperature at 30-45°C would not affect histological and immunohistochemical analysis. Comparison was made with traditional decalcification method with regards to quality of tissue morphology and antigenicity.
A fresh human cadaveric femoral shaft was sectioned into 5-mm-thick transverse sections. After fixation, the bone slices were divided into two groups: Ultrasound decalcification group (US DeCal), in which bone sections (n = 3) were placed in a US decalcifier (50 W at a frequency of 40kHz) with EDTA solution, and normal decalcification group (Normal DeCal), in which bone sections (n = 3) were decalcified in EDTA without US. The mineral content of the bone sections was measured with micro-computed tomography and dual-energy X-ray absorptiometry at different time points. Rate of calcium extraction was quantified by measuring the calcium concentration in EDTA solution using inductively coupled plasma optical emission spectrometry. After decalcification, the paraffin sections of the decalcified bone were stained with haematoxylin and eosin or immunohistochemical staining of sclerostin.
Samples in US DeCal contained 2.9 ± 2.8% of the mineral content at Day 6 and were completely decalcified at Day 8. However, sections in Normal DeCal retained 36.3 ± 5.1% and 24.3 ± 4.8% at Day 6 and Day 8, respectively, and took six times longer to complete decalcification. The concentration of calcium in the EDTA solution of the US DeCal group was 70% higher than that of the Normal DeCal group (p < 0.05) in Day 1 and 2. No staining difference was observed in histological sections between the two groups.
The application of US decalcification significantly shortened the decalcification time in EDTA without causing histological artefacts.
This article shows that the application of ultrasound in sample decalcification would shorten the duration that decalcification required. This would accelerate the sample processing for routine bone histology in both basic and clinical research and assessments for diagnostic purposes.
背景/目的：骨标本脱钙是常规石蜡包埋和切片所必需的。乙二胺四乙酸（EDTA）作为一种脱钙螯合剂，可保持骨组织的完整性和组织学特征，但脱钙时间长，尤其是对于矿化基质致密的皮质骨。我们假设，一种新上市的超声脱钙剂能有效加速EDTA中厚皮质骨标本的脱钙，且30 - 45°C的工作温度不会影响组织学和免疫组织化学分析。在组织形态质量和抗原性方面，将其与传统脱钙方法进行比较。
将新鲜人尸体股骨干切成5毫米厚的横切片。固定后，将骨切片分为两组：超声脱钙组（US DeCal），将骨切片（n = 3）置于含有EDTA溶液的超声脱钙剂（功率50 W，频率40kHz）中；常规脱钙组（Normal DeCal），将骨切片（n = 3）置于不含超声的EDTA中脱钙。在不同时间点，用微型计算机断层扫描和双能X线吸收法测量骨切片的矿物质含量。通过电感耦合等离子体发射光谱法测量EDTA溶液中的钙浓度，定量钙提取率。脱钙后，对脱钙骨的石蜡切片进行苏木精和伊红染色或硬化蛋白免疫组织化学染色。
超声脱钙组样本在第6天矿物质含量为2.9±2.8%，并在第8天完全脱钙。然而，常规脱钙组切片在第6天和第8天分别保留36.3±5.1%和24.3±4.8%，脱钙所需时间长6倍。在第1天和第2天，超声脱钙组EDTA溶液中的钙浓度比常规脱钙组高70%（p < 0.05）。两组组织学切片未观察到染色差异。
超声脱钙的应用显著缩短了EDTA中的脱钙时间，且未造成组织学假象。
本文表明超声在样本脱钙中的应用将缩短脱钙所需时间。这将加快基础和临床研究中常规骨组织学样本处理以及诊断目的评估。
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