Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis
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, espe...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2019-04-01
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| Series: | Journal of Orthopaedic Translation |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214031X18300962 |
| _version_ | 1819260617279668224 |
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| author | Dick H. Chow Lizhen Zheng Li Tian Kam-Sing Ho Ling Qin Xia Guo |
| author_facet | Dick H. Chow Lizhen Zheng Li Tian Kam-Sing Ho Ling Qin Xia Guo |
| author_sort | Dick H. Chow |
| collection | DOAJ |
| description | 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 |
| first_indexed | 2024-12-23T19:28:45Z |
| format | Article |
| id | doaj.art-e3cd2629fad94cf9986a4d634c86b8d7 |
| institution | Directory Open Access Journal |
| issn | 2214-031X |
| language | English |
| last_indexed | 2024-12-23T19:28:45Z |
| publishDate | 2019-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Orthopaedic Translation |
| spelling | doaj.art-e3cd2629fad94cf9986a4d634c86b8d72022-12-21T17:33:59ZengElsevierJournal of Orthopaedic Translation2214-031X2019-04-0117112120Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysisDick H. Chow0Lizhen Zheng1Li Tian2Kam-Sing Ho3Ling Qin4Xia Guo5Musculoskeletal 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 RegionMusculoskeletal 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 RegionMusculoskeletal 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 RegionMusculoskeletal 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 RegionMusculoskeletal 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 RegionDepartment of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region; Corresponding author. Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hung Hom, Hong Kong Special Administrative Region.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, Ultrasoundhttp://www.sciencedirect.com/science/article/pii/S2214031X18300962 |
| spellingShingle | Dick H. Chow Lizhen Zheng Li Tian Kam-Sing Ho Ling Qin Xia Guo Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis Journal of Orthopaedic Translation |
| title | Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis |
| title_full | Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis |
| title_fullStr | Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis |
| title_full_unstemmed | Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis |
| title_short | Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis |
| title_sort | application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis |
| url | http://www.sciencedirect.com/science/article/pii/S2214031X18300962 |
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