Application of Ultrasound Accelerates the Decalcification of Human Bone Sample for Histological Analysis
Refereed conference paper presented and published in conference proceedings
CUHK Authors
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AbstractINTRODUCTION: The inorganic phase of bone is composed of calcium hydroxyapatite, Ca10(PO4)6(OH)2, and accounts for 60% to 70% of the tissue [1]. Decalcification is a process to soften the bone specimen by dissolution of the hydroxyapatite complex in bone matrix. After decalcification, the bone samples would be compatible with routine paraffin histologic preparation. However, the traditional decalcification method requires long incubation time to complete this process. Ultrasound (US) is believed to enhance the decalcification by a cavitation mechanism [2]. We hypothesized that the application of US during bone decalcification would accelerate the decalcification compare with the traditional decalcification method without damaging bone tissue for histological assessments.
METHODS: A piece of human femur was obtained from Science Care (Arizona, USA). The femoral shaft was sectioned into 5mm thick transverse sections. The bone slices (n=6) were fixed in 4% phosphate buffered paraformaldehyde for 24 hours and divided into two groups: Ultrasound Decalcification group (US DeCal) and Normal Decalcification group (Normal DeCal). For US DeCal, the bone sections were place in the US decalcifier (DeCa DX100, Pro-Cure Medical Technology Co. Ltd, Hong Kong, 50W at a frequency of 40kHz) with 300mL of 0.5M EDTA solution and maintained at 30 - 45°C. For the Normal DeCal, bone slices were placed in a container with 300mL of 0.5M EDTA and maintained at 37°C. The EDTA solution was refreshed daily. The mineral content of the bone slices was measured by micro-CT and Dual-energy X-ray absorptiometry (DXA) at different time points. Calcium concentration of EDTA was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). After decalcification was completed, the samples were processed, embedded in paraffin, sectioned at 5μm, and stained with hematoxylin and eosin (H&E) for assessment.
RESULTS SECTION: The samples from US DeCal contained less than 7% of the mineral content at Day 6 and they were completely decalcified at Day 8 (Figure 1). The samples in Normal DeCal retained 36.32±5.09% and 24.30±4.80% at Day 6 and Day 8 respectively (Figure 2a, 2b, 2c, and 2d). Normal DeCal samples took 50 Days to complete decalcification. The concentration of calcium ions in the EDTA solution of the US DeCal group was 80% higher than the Normal DeCal (p<0.05, Figure 2e). There was no significant difference between the sections of the two groups histologically (Figure 3).
DISCUSSION: Our results suggested that decalcification by US shorten the duration by six times compare with the normal decalcification. Others reported that US shortened the decalcification process by 4-12 times depending on the thickness of the sample and the type of tissues [3, 4].
SIGNIFICANCE: Ultrasound decalcification enhanced the rate of decalcification of bone slices compared with normal decalcification method. The application of our technology would accelerate the routine sample decalcification process for bone histology in both basic and clinical research.
REFERENCES
1. Einhorn T.A. et al. Orthopaedic basic science : biology and biomechanics of the musculoskeletal system. 2nd ed2000, Rosemont, Ill.: American Academy of Orthopaedic Surgeons. xix, 873 p.
2. Leung K. et al. A practical manual for musculoskeletal research, 2008, Singapore: World Scientific.
3. Milan L. et al., Am J Surg Pathol, 1981. 5(6): p. 573-9.
4. Thorpe E.J. et al., J Bone Joint Surg Am, 1963. 45: p. 1257-9.
ACKNOWLEDGEMENTS: This project was supported by the Public Sector Trial Scheme in the Innovation and Technology Commission of the Hong Kong SAR (Ref. ITT/003/13GP) and SMART Program, Lui Che Woo Institute of Innovative Medicine, Faculty of Medicine, the Chinese University of Hong Kong supported by Lui Che Woo Foundation Limited.
METHODS: A piece of human femur was obtained from Science Care (Arizona, USA). The femoral shaft was sectioned into 5mm thick transverse sections. The bone slices (n=6) were fixed in 4% phosphate buffered paraformaldehyde for 24 hours and divided into two groups: Ultrasound Decalcification group (US DeCal) and Normal Decalcification group (Normal DeCal). For US DeCal, the bone sections were place in the US decalcifier (DeCa DX100, Pro-Cure Medical Technology Co. Ltd, Hong Kong, 50W at a frequency of 40kHz) with 300mL of 0.5M EDTA solution and maintained at 30 - 45°C. For the Normal DeCal, bone slices were placed in a container with 300mL of 0.5M EDTA and maintained at 37°C. The EDTA solution was refreshed daily. The mineral content of the bone slices was measured by micro-CT and Dual-energy X-ray absorptiometry (DXA) at different time points. Calcium concentration of EDTA was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). After decalcification was completed, the samples were processed, embedded in paraffin, sectioned at 5μm, and stained with hematoxylin and eosin (H&E) for assessment.
RESULTS SECTION: The samples from US DeCal contained less than 7% of the mineral content at Day 6 and they were completely decalcified at Day 8 (Figure 1). The samples in Normal DeCal retained 36.32±5.09% and 24.30±4.80% at Day 6 and Day 8 respectively (Figure 2a, 2b, 2c, and 2d). Normal DeCal samples took 50 Days to complete decalcification. The concentration of calcium ions in the EDTA solution of the US DeCal group was 80% higher than the Normal DeCal (p<0.05, Figure 2e). There was no significant difference between the sections of the two groups histologically (Figure 3).
DISCUSSION: Our results suggested that decalcification by US shorten the duration by six times compare with the normal decalcification. Others reported that US shortened the decalcification process by 4-12 times depending on the thickness of the sample and the type of tissues [3, 4].
SIGNIFICANCE: Ultrasound decalcification enhanced the rate of decalcification of bone slices compared with normal decalcification method. The application of our technology would accelerate the routine sample decalcification process for bone histology in both basic and clinical research.
REFERENCES
1. Einhorn T.A. et al. Orthopaedic basic science : biology and biomechanics of the musculoskeletal system. 2nd ed2000, Rosemont, Ill.: American Academy of Orthopaedic Surgeons. xix, 873 p.
2. Leung K. et al. A practical manual for musculoskeletal research, 2008, Singapore: World Scientific.
3. Milan L. et al., Am J Surg Pathol, 1981. 5(6): p. 573-9.
4. Thorpe E.J. et al., J Bone Joint Surg Am, 1963. 45: p. 1257-9.
ACKNOWLEDGEMENTS: This project was supported by the Public Sector Trial Scheme in the Innovation and Technology Commission of the Hong Kong SAR (Ref. ITT/003/13GP) and SMART Program, Lui Che Woo Institute of Innovative Medicine, Faculty of Medicine, the Chinese University of Hong Kong supported by Lui Che Woo Foundation Limited.
All Author(s) ListDick H. Chow, Li-Zhen Zheng, Li Tian, Kam-Sing Ho, Ling Qin, Xia Guo
Name of ConferenceOrthopaedic Research Society 2016 Annual Meeting
Start Date of Conference04/03/2016
End Date of Conference08/03/2016
Place of ConferenceOrlando, Florida
Country/Region of ConferenceUnited States of America
Year2016
Month3
LanguagesEnglish-United States
