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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 2  |  Page : 94-100

A quantitative study of comparing the routine decalcification and microwave decalcification methods by using different decalcifying agents


1 Department of Oral Pathology & Microbiology, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed University, Maharashtra, India
2 Department of Public Health Dentistry, School of Dental Sciences, Krishna Institute of Medical Sciences Deemed University, Maharashtra, India
3 Private Dental practioner Karad, Maharashtra, India

Date of Web Publication28-Dec-2017

Correspondence Address:
Dr. Vidya Kadashetti
School of Dental Sciences, Krishna Institute of Medical Sciences Deemed University, Malkapur, Karad, Satara - 415 110, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmd.ijmd_37_17

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  Abstract 


Background: Decalcification is a process of complete removal of calcium salts from mineralized tissues. Microwave decalcification is a novel technique that can accelerate the duration of decalcification compared to the manual method, by increasing the temperature to evaluate the fastest decalcifying agents and methods (routine with Microwave decalcification) in both bone and tooth.
Materials and Methods: The study group consisted of 80 hard tissues, 40 teeth and 40 pieces of 1 cm × 1 cm cut bone specimens were decalcified by both routine and microwave method using four decalcifying solutions (five bone pieces and five teeth in each agents for both routine and microwave method). The four decalcifying solutions were 8% nitric acid, 8% formic acid, 8%formal nitric acid, and 14% of ethylenediaminetetraacetic acid (EDTA).
Results: Duration of decalcifi cation of bone and tooth by both methods with 8% nitric acid took 10 days for CD and 2 days for MD respectively. Similarly for rest of the acids took more time to decalcify in CD method than MD method. Fourteen percent EDTA gave excellent results followed by 8% formal nitric acid, and 8% formic acid has good results than 8% nitric acid.
Conclusion: Microwave processing proved to be an efficient and reliable procedure for the decalcification of bones and tooth. Microwave decalcification yielded quicker and better results compared to the routine methods. Therefore, microwave can serve as a quicker and reliable diagnostic method for a pathologist.

Keywords: Bone; decalcification; microwave; routine; teeth


How to cite this article:
Kadashetti V, Shivakumar K M, Baad R, Vibhute N, Belgaumi U, Bommanavar S, Kamate W, Sankapal S. A quantitative study of comparing the routine decalcification and microwave decalcification methods by using different decalcifying agents. Indian J Multidiscip Dent 2017;7:94-100

How to cite this URL:
Kadashetti V, Shivakumar K M, Baad R, Vibhute N, Belgaumi U, Bommanavar S, Kamate W, Sankapal S. A quantitative study of comparing the routine decalcification and microwave decalcification methods by using different decalcifying agents. Indian J Multidiscip Dent [serial online] 2017 [cited 2019 Jul 21];7:94-100. Available from: http://www.ijmdent.com/text.asp?2017/7/2/94/221764




  Introduction Top


The physical hardness, which is, a unique characteristic of these tissues makes it necessary to “soften” them by removing the mineralized component. Histological observations of the hard tissue require the removal of the mineral component.[1] The head and neck is a complex structure of both soft and hard tissues. Most of the soft tissues need not require any prior treatment before tissue processing and put forth little resistance to the histochemical techniques. Lesions of hard tissues need intricate, technique-sensitive methodology for interpretation and quicker diagnosis. Thin sections of teeth also provide an excellent tool for dental research.[2] Histological research of the bone, tooth pulp, immature enamel, dentin, and cementum requires the removal of the mineral component from the bone, dentin, and cementum, and for enamel will not be seen in decalcified sections because it is made up of 96% of inorganic, and after decalcification, there will be no retaining of any structure of enamel.[3] The sectioning of these hard tissues by ordinary methods is impossible. Such tissues must be treated to remove mineral content by a process known as “decalcification” or “demineralization” thereby preparing the tissue soft enough to be sectioned by the microtome.[4] Decalcification is a process of complete removal of calcium salts from mineralized tissues. The physical hardness, which is, a unique characteristic of these tissues makes it necessary to “soften” them by removing the calcium salts.[3] There are other methods to look for anatomy of teeth, and depending on them, various types of sectioning is adopted such as ground sections. Routine methods to study the histology of oral tissues involve preparation of tissue, sectioning, staining, and then observing under microscope; overall procedure is tissue processing. It is for both hard or soft tissue necessary to keep it in a life-like manner and to impregnate with a solid medium, so it will give enough rigidity to facilitate the preparation of thin sectioning for study under microscope [2]

Hard tissue decalcification is one of the most technique sensitive procedures in the histopathology laboratory. It is of more significance in oral pathology laboratory as decalcification of bone and teeth is a routinely required procedure.[2] Removal of calcium salts is carried out by certain chemical agents, either with different grades of acids to form soluble calcium salts or chelating agents that bind to calcium ions.[5] Hard tissue specimens differ from their soft tissue counterpart, in that decalcification done before routine processing and staining. The end point of decalcification is determined using various methods including routine radiograph and chemical tests. At present, the average time taken for Conventional Decalcification (CD) is 3–6 days (depending on the size of the specimen and type of acid used). For quick and reliable diagnosis, the demand for faster decalcification is ever growing, especially in cases of malignancies wherein the urgent diagnosis is required.[6] Factors affecting the decalcifications are type of decalcifying agent, concentration of acid, pH, and temperature. Microwave decalcification is a novel technique that can accelerate the duration of decalcification compared to the manual method, by increasing the temperature. This method speed up the process of decalcification significantly from days to hours.[5] Vongsavan N, et al (1990) have reported that the duration of decalcification is accelerated about 10 times compared with that at ambient temperature.[7] The aims and objectives of the study were as follows:

  1. To evaluate the fastest decalcifying agents and methods (routine with microwave decalcification) in both bone and tooth
  2. To evaluate the decalcifying agents based on its effect on the organic content, integrity of the soft tissues and staining characteristics of tooth and bone by both methods
  3. To compare and contrast between the decalcifying agents and methods.



  Materials and Methods Top


The study group consisted of 80 hard tissues, 40 healthy single-rooted tooth specimen, and 40 equal sizes (1 cm × 1 cm) cut sections of radius bone specimens. The specimens were fixed in 10% buffered formalin. The study was performed after obtaining clearance from the ethical board of the institution. Tooth was extracted for orthodontic reasons or any mobility reason, and bone specimens from the archives of the Department of General Pathology were used in the study. A total of 40 teeth and 40 pieces of 1 cm × 1 cm cut bone specimens were decalcified by both routine and microwave method using four decalcifying solutions (five bone pieces and five teeth in each agents for both routine and microwave method). The four decalcifying solutions were 8% nitric acid, 8% formic acid, 8% formal nitric acid, and 14% of ethylenediaminetetraacetic acid (EDTA). These solutions were chosen as they are commonly used and easily available. Inclusion criteria are single-rooted healthy teeth specimen indicated for extraction for orthodontic purpose or any mobility reason, radius bone specimens (indicated for resection was collected postdiagnosis). Bone specimens were sectioned into 40 pieces of uniform size (approximately 1 by 1 cm). Exclusion criteria are teeth with caries and restorations, malignant bone specimens, or any other pathology of bone.

For conventional decalcification (CD): The formalin-fixed hard tissue specimens were water washed for 30 min before being placed in decalcifying agents at room temperature. The time and date was noted to compare the duration of decalcification. The solutions were changed periodically till complete decalcification was achieved. The end point of decalcification was confirmed with a radiograph as it provides an accurate interpretation of the same without damaging the tissue specimen.

For a domestic microwave oven (BAJAJ, Model 2310ETC) with a fixed rotary plate was used. A Coplin jar containing 100 ml of distilled water was preheated for 5 s to warm up the magnetron. All the specimens were fixed in 10% neutral-buffered formalin fixative and then washed in water for about 30 min before decalcification. All the specimens were labeled for standardization of procedure. Each sample was placed in Coplin jar with approximately 100 ml of decalcifying agent for decalcification. The exact time at the start of decalcification was noted. The pH and temperature of the solutions were recorded; decalcifying solutions were changed on a daily basis. In the microwave technique, the specimens were irradiated for eight cycles of 10 s each (at 1 h intervals) per day for all decalcifying agents used in the study. The end point of decalcification was confirmed with a radiograph as it provides an accurate interpretation of the same without damaging the tissue specimen. The microwave decalcification protocol employed in the present study was based on a modification of the methodology employed by Sangeetha et al.[4] Following the decalcification, the specimens were subjected to routine processing and staining.

Tissue processing and staining

After ensuring complete decalcification, the tissues were washed using distilled water for 30 min, following which the all specimens were subjected to manual tissue processing. After processing, the tissues were embedded in paraffin and were sectioned to a thickness of 7–8 μm using the soft tissue microtome. The sections were then stained by Harris' hematoxylin and eosin Y. The stained sections of decalcified bone and teeth were assessed for the quality of staining and preservation of tissue details as objectively as possible. The following parameters were recorded for each specimen:

For bone specimens,

  • Missing osteocytes from the lacunae.


For teeth,

  • Shrinkage of pulp away from the dentinal wall
  • Damage to odontoblastic layer.


For both,

  • Yellow discoloration of specimen
  • Patchy staining.


The data obtained from the above observations were entered and analyzed for results The stained sections were observed under the microscope and were graded from 1 to 4 (1 – poor, 2 – fair, 3 – good, and 4 – excellent) based on the following criteria:

  1. Ease of sectioning
  2. Hard-tissue staining
  3. Soft-tissue staining – both cytoplasmic and nuclear
  4. Staining
  5. Soft-tissue attachment and
  6. Pulpal organization.



  Results Top


[Graph 1] shows the comparison of duration of decalcification of bone by both methods with different decalcifying agents; 8% nitric acid took 10 days for CD and for MD 2 days, 8% formic acid took 14 days CD and for MD 5 days, 8% formal nitric acid took12 days for CD and for MD 4 days, and 14% EDTA has taken 64 days for CD and for MD 12 days to decalcify the bone.



[Graph 2] shows the comparison of duration of decalcification of tooth by both methods with different decalcifying agents; 8% nitric acid took 10 days for CD and for MD 3 days, 8% formic acid took 16 days CD and for MD 6 days, 8% formal nitric acid took14 days for CD and for MD 5 days, and 14% EDTA has taken 70 days for CD and for MD 24 days to decalcify the tooth.



[Graph 3] and [Graph 4] show the evaluation the decalcifying agents based on its effect on the organic content and integrity of the soft tissue in tooth by conventional method and microwave method. Fourteen percent EDTA gave excellent results followed by 8% formal nitric acid, and 8% formic acid has good results than 8% nitric acid.



[Table 1] summarizes the cumulative scores of the decalcifying agents based on the various parameters 14 EDTA has excellent results in both CD and MD method of decalcification compared to the other decalcifying agents.
Table 1: Cumulative scores of the decalcifying agents based on various parameters

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  Discussion Top


In contrast to soft-tissue specimens, hard-tissue specimens have to be decalcified before processing and staining. Pathologists are under constant pressure for instant and reliable diagnosis. Thus, the present study was aimed to evaluate and compare the conventional decalcification and microwave decalcification with different decalcifying agents.

The most commonly employed decalcifying agents include nitric acid, formic acid, formal nitric acid, and EDTA. Among these, decalcifying agents except EDTA are known to be comparatively more corroding to the tissues at higher concentration. Increasing the concentration of the decalcifying agent should decrease the decalcifying time, if increased beyond a certain concentration, it will result in significant tissue damage and if reduced, the concentration may fall short in its inability to produce results at a faster rate.[2],[3],[4] Thus, a balance to be achieved between the time taken for decalcification and the output of the final specimen. Depending on the depth of investigation necessary, the pathologist must select the agent of choice and customize the concentration of the decalcifying agent. Other factor like temperature has proven to have significant effect on the decalcification time and the final quality of tissue sections. Increasing the temperature of the decalcifying agent will increase the rate of penetration of the agent into the specimen.[4],[6],[8] Microwave produces uniform increase in temperature throughout the specimen. Thus, microwave decalcification could hasten the diagnostic time without compromising the diagnostic quality of the tissue.[4],[9],[10]

The energy produced by microwaves generated in a domestic oven interacts with dipolar molecules by imparting kinetic energy and altering the electric fields. This energy induces a dielectric field leading to a rapid oscillation of dipolar molecules at about 180°C, generating heat, that is, rapidly distributed homogeneously within the tissue. Pitol et al. showed that there was a 30-fold increase in decalcification speed compared to the traditional method when the material was irradiated in a microwave oven.[11] However, Vongsavan N, et al (1990) reported that the decalcification of bone is accelerated about 10 times in the microwave oven compared with that at ambient temperature.[7] In our study, the 14% of EDTA reagent took 64 days in conventional method (CD) and 12 days for Microwave method (MD) and 8% nitric acid took 10 days for CD and 2 days for MD. In the present study, the 14% of EDTA reagent took 70 days in conventional method (CD) and 24 days for Microwave method (MD) and 8% nitric acid took 10 days for CD and 2 days for MD; similarly, 8% formic acid and 8% formal nitric acid took longer duration for conventional method and less time for microwave method to decal the bone and tooth specimen [Graph 1] and [Graph 2]. Microwave method reduced the decalcification time for both the bone and tooth specimens by one fourth in comparison to conventional method. Similar results were obtained from the study conducted by Pitol et al. and Sangeeta et al.[4],[11]

In terms of efficacy of agents with respect to soft-tissue integrity and hard- and soft-tissue staining, nuclear staining, and cytoplasmic staining, excellent results were obtained with the slowest decalcifying agent, i.e., 14% EDTA, and rest of the acids showed average results when compared with EDTA in conventional method and by microwave method also [Figure 1] and [Figure 2]. These results were in agreement with study conducted by Sanjai et al.[2] The present study showed that overall impression on decalcification by microwave method is excellent even though by using different decalcifying agents, even for 14% EDTA microwave method accelerate the decalcification because it is one slowest decalcifying agent. In the present study, when we compared among different decalcifying agents, 14% EDTA was excellent in staining, nuclear details, and cytoplasmic details when compared to other agents but duration of decalcification was slow compared to other agents. In the study conducted by Waerhaug, decalcification of bone and teeth was under vacuum. The time taken for decalcification was reduced to one-tenth.[12] Increase in temperature at which decalcification occurs also varies the time taken for complete decalcification. In a study by Vongsavan et al., on cat and rat teeth, decalcification will be faster in microwave than conventional method.[7] The study by Pitol et al. showed that decalcification by microwave showed to be more effective than the conventional methods in aspects such as reduction duration for decalcification, morphology of bone tissue, and an increase of calcium release using microwave.[11] In our study is concordance In In our study it is in concordance with these results and also states that the decalcifying acids can be used in microwave method which gives good staining quality that will be helpful for urgent hard-tissue reporting and the staining details will be more better in 14%EDTA [Figure 1], [Figure 2], [Figure 3], [Figure 4]. If time is not factor, 14% EDTA can be used for preservation, presentation, excellent soft-tissue integrity, and quality of staining. In literature search we found using different decalcifying agents, namely, nitric acid, formic acid, and EDTA, more studies found using EDTA than other decalcifying agents. Comparison between acidic agents formal nitric acid gives good results than nitric acid and formic acid, i.e., in duration as well as soft-tissue integrity and quality of staining [Table 1] and [Figure 5], [Figure 6], [Figure 7], [Figure 8].
Figure 1: Tooth decalcified using 14% ethylenediaminetetraacetic acid by microwave decalcification H and E staining

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Figure 2: Bone decalcified using 14% ethylenediaminetetraacetic acid by microwave decalcification H and E staining

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Figure 3: Tooth decalcified using 14% ethylenediaminetetraacetic acid by routine decalcification by H and E staining

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Figure 4: Bone decalcified using 14% ethylenediaminetetraacetic acid by routine decalcification by H and E staining

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Figure 5: Tooth decalcified using 8% formal nitric acid by routine decalcification by H and E staining

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Figure 6: Bone decalcified using 8% formal nitric acid by routine decalcification by H and E staining

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Figure 7: Tooth decalcified using 8% formal nitric acid by microwave decalcification by H and E staining

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Figure 8: Bone decalcified using 8% formal nitric acid by microwave decalcification by H and E staining

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  Conclusion Top


In cases of urgent requirement for reporting of hard tissue histopathology, acids can be used, and accelerating decalcification microwave method will be more useful than conventional method. Hard the structure, hardest to decalcify. For preservation and presentation, EDTA can be used. In EDTA, decalcification slower the process, better the results. Microwave processing proved to be an efficient and reliable procedure for the decalcification of bones and tooth. Microwave decalcification yielded quicker and better results compared to the routine methods. Therefore, microwave can serve as a quicker and reliable diagnostic method for a pathologist.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Cook SF, Ezra-Cohn HE. A comparison of methods for decalcifying bone. J Histochem Cytochem 1962;10:560-3.  Back to cited text no. 1
    
2.
Sanjai K, Kumarswamy J, Patil A, Papaiah L, Jayaram S, Krishnan L, et al. Evaluation and comparison of decalcification agents on the human teeth. J Oral Maxillofac Pathol 2012;16:222-7.  Back to cited text no. 2
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3.
Prasad P, Donoghue M. A comparative study of various decalcification techniques. Indian J Dent Res 2013;24:302-8.  Back to cited text no. 3
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4.
Sangeetha R, Uma K, Chandavarkar V. Comparison of routine decalcification methods with microwave decalcification of bone and teeth. J Oral Maxillofac Pathol 2013;17:386-91.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Roncaroli F, Mussa B, Bussolati G. Microwave oven for improved tissue fixation and decalcification. Pathologica 1991;83:307-10.  Back to cited text no. 5
    
6.
Raj AT, Patil S, Rao RS. A comparison of conventional and microwave decalcification and processing of tooth and mandibular bone specimens. J Clin Diagn Res 2016;10:ZC121-6.  Back to cited text no. 6
    
7.
Vongsavan N, Matthews B, Harrison GK. Decalcification of teeth in a microwave oven. Histochem J 1990;22:377-80.  Back to cited text no. 7
    
8.
Bancroft JD, Gamble M. Theory and Practice of Histological Technique. 7th ed., Ch. 16. Philadelphia, PA: Churchill Livingstone Elsevier; 2013. p. 324-8.  Back to cited text no. 8
    
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Amrutha N, Patil S, Rao RS. Microwaves: A revolution in histoprocessing. J Contemp Dent Pract 2014;15:149-52.  Back to cited text no. 9
    
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Babu TM, Malathi N, Magesh KT. A comparative study on microwave and routine tissue processing. Indian J Dent Res 2011;22:50-5.  Back to cited text no. 10
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11.
Pitol DL, Caetano FH, Lunardi LO. Microwave-induced fast decalcification of rat bone for electron microscopic analysis: An ultrastructural and cytochemical study. Braz Dent J 2007;18:153-7.  Back to cited text no. 11
    
12.
Waerhaug J. Decalcification of bone and teeth under vacuum; a rapid method for producing hard tissue preparations. J Dent Res 1949;28:525.  Back to cited text no. 12
    


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