Driving the Mineral out Faster: Simple Modifications of the Decalcification Technique

Kapila, Supriya Nikita; Natarajan, Srikant; Boaz, Karen; Pandya, Jay Ashokkumar; Yinti, Shanmukha Raviteja

doi:10.7860/jcdr/2015/14641.6569

Cited by 12 publications

(16 citation statements)

References 6 publications

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“…Different methods, such as modifying the decalcification conditions including higher concentration of acid and increasing the temperature of the decalcifying agent, have been demonstrated to accelerate and shorten decalcification. However, these methods may damage the tissue integrity and introduce artefacts to the samples, such as osteocyte retraction [3].…”

Section: Discussionmentioning

confidence: 99%

“…The stained sections of both the groups were then observed under a light microscope for assessment of cell morphology and to test whether any artefacts or detrimental effects were introduced into the tissues during the decalcification. The ratio of cells with nucleus to total number of cells was measured within a region of interest of mm by mm at a 200× magnification to determine whether the samples had been over decalcified because over decalcification would cause problems with nuclear staining and lead to osteocyte retraction in bone sections [3], [5].…”

Section: Methodsmentioning

confidence: 99%

“…However, the traditional decalcification method requires long incubation time for this process to get completed. The duration of the decalcification process is dependent on several factors including the thickness of the sample, type and concentration of decalcifying agent, the temperature at which the decalcification process takes place and the presence or absence of any mechanical agitation such as shaking and stirring [3].…”

Section: Introductionmentioning

confidence: 99%

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

Chow

Zheng

Tian

et al. 2019

Journal of Orthopaedic Translation

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Chow

Zheng

Tian

et al. 2019

Journal of Orthopaedic Translation

View full text Add to dashboard Cite

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“…Rising the temperature can accelerate the decalcification speed (21), but there are some concerns about the risk of overheating; and temperature above 45°C is not recommend (10,22). Mechanical agitation was demonstrated to accelerate decalcification by replacing the calcium saturated solutions surrounding the samples with fresh mediums (10,21,22), and slow stirring agitation represented better histologic results (22). In the current study, the procedure was performed at room temperature due to lack of instruments that prevent overheating.…”

Section: Discussionmentioning

confidence: 99%

Preparation of Human Inner Ear Structures for High Resolution Imaging Studies

et al. 2017

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Background: Introduction of the novel imaging modalities such as optical coherence tomography and scanning laser optical tomography allow the morphological and functional evaluation of the intracochlear structures up to the histological details. The prerequisite of these modalities is the preparation of the membranous labyrinth by removing the dense otic capsule to enhance the optical penetration depth. In the present study, a combination of the chemical decalcification and bone drilling method was explained for the preparation of the human inner ear structures for further studies. Methods: In this study, nine human temporal bones were used and trimmed in cubes containing middle and inner ear structures. The samples were immersed in 5% nitric acid, 20% EDTA, and 10% EDTA solutions, respectively. The samples were brought out and rinsed every 90 minutes, and mechanical removal of the softened bone was performed with surgical drill system until the complete decalcification of the samples was confirmed by X-ray imaging. The prepared samples were evaluated by microcomputed tomography imaging for anatomical distortions caused by the preparation process.

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“…Some typical decalcifying agents include, nitric acid, Gooding and Stewart's fluid, Rapid Bone Decalcifier (RDO), and chelating agents. More recently, new methods have been discovered to allow hard tissues to be decalcified faster[23].…”

mentioning

confidence: 99%