Conclusion
The application of US decalcification significantly shortened the decalcification time in EDTA without causing histological artefacts. The translational potential of this article: 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.
Methods
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.
Results
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.