Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis

Abstract

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.

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.

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.

Keywords: Bone histology; Bone histomorphometry; Decalcification; Immunohistochemistry; Ultrasound.

Figure 1

Representative bone slices of Normal DeCal and US DeCal after complete decalcification, without showing significant difference in appearance. Scale bar: 10mm.

Figure 2

Measurements of (A) BMC , (B) BMD , (C) BV (D) and BV/TV Normal decalcification and US decalcification group. * = p < 0.01; ** = p < 0.001.

Figure 3

Three-dimensional images of the bone slices over time. US DeCal showed significantly faster decrease in the mineralised tissue than Normal DeCal. The central core of the bone slice in the Normal DeCal group remained calcified at Day 8, whereas the tissue of outer layer had been completely decalcified in the US DeCal group. Scale bar: 10mm.

Figure 4

Concentration of calcium ions in the decalcification solution was measured by ICP-OES. The calcium concentration was significantly higher in the US DeCal group than in the Normal DeCal group by 80% at Day 1 and 2 of decalcification. ** = p < 0.001; * = p < 0.05.

Figure 5

Correlation between Ca²⁺ concentration in EDTA solution and mineralised tissue. Positive correlations were observed between Ca²⁺ concentration and BMC, BMD, BV and BV/TV. Regarding the linear line of best fit, the lines of the US DeCal and Normal DeCal had comparable slopes. Furthermore, the lines of the US DeCal were shifted to the left compared with those of the Normal DeCal.

Figure 6

(A) Representative H&E images of samples from US DeCal and Normal DeCal groups. The transverse sections showed numerous osteons with peripheral cement line. Well-stained osteocyte nuclei indicate that the decalcification endpoint was not exceeded. There is no significant difference between the two groups with respect to the histological features and presence of artefacts; (B) The ratio of the osteocytes with identifiable nucleus to the total number of osteocytes did not show any significant difference between US DeCal and Normal DeCal groups. Magnification: 200×. Scale Bar: 200μm.

Figure 7

Expression and presence of (A) sclerostin and (B) osteocalcin in US DeCal and Normal DeCal samples at different magnifications (50× and 200×). No significant difference was observed between the two groups. Red dotted box: Zoom-in area of 200× image. Black scale bar: 500μm; white scale bar: 100μm.

Figure 8

The ratio of sclerostin (Scl)-positive cells to total number of cells between US DeCal and Normal DeCal. There was no significant difference between the two groups.