| Abstract|| |
Background : Study of fibrilar, cellular and sub cellular structures of mineralized tissues is only possible after the removal of the calcium apatite of these tissues by the process of demineralization.
Aims: The present study aims to evaluate six commonly used demineralizing agents to identify the best decalcifying agent.
Materials and Methods: The present study included six different decalcifying solutions: 10% formal nitric acid, 8% formal nitric acid, 10% formic acid, 8% formic acid, Perenyi's fluid and Ethylene Di-Amine Tetra Acetic Acid. eight samples of posterior mandible of rat were decalcified in each of the decalcifying solutions and subjected to chemical end-point test. Ehrlich's Hematoxylin stain was used.
Statistical Analysis Used: One way ANOVA was used for multiple group comparisons and Chi-square test was used for analyzing categorical data. P value of 0.05/less was set for statistical significance.
Results: Samples treated with EDTA showed the best overall histological impression and the tissue integrity were well preserved. Formal nitric of both the percentages 10 and 8% gave fairly good cellular detail and were rapid in their action.
Conclusion: The final impression led to the proposition that EDTA was indeed the best decalcifying agent available. However, with time constraint, the use of formal nitric acid is advocated.
Keywords: 10% formal nitric acid, 10% formic acid, 8% formal nitric acid, 8% formic acid, decalcification, Ehrlich′s stain, Perenyi′s fluid and Ethylene Di-Amine Tetra Acetic Acid, tooth and bone
|How to cite this article:|
Prasad P, Donoghue M. A comparative study of various decalcification techniques. Indian J Dent Res 2013;24:302-8
Decalcification is a process of complete removal of calcium salt from mineralized tissues like bone and teeth and other calcified tissues. The physical hardness, which is, a unique characteristic of these tissues makes it necessary to "soften" them by removing the mineralized component. Human dentition and bone has been the subject of intense histological investigations for many years. Histological observations of the pulp, immature enamel, dentin and cementum, require the removal of the mineral component of the dentin and cementum.  Dental pulp evaluation is often a part of research protocols followed in assessment of pulpal biological response to new restorative materials. Examination of demineralized sections of other dental tissues and bone is necessary to study various pathological and developmental processes. Additionally various other soft tissues may be hardened and need decalcification following alterations due to dystrophic and metastatic processes. In all these cases, the histopathologist can provide hard tissue biopsy reports only after the process of decalcification.
Demineralization or decalcification of tissues is a routine process carried out in most laboratories by the use of various acids or chelating agents. However, it is an inherently complex process, complicated by the tradeoff between time taken for the process and the quality of the sections produced. Where the stronger acids provide the fastest result with the poorest quality of sections, chelating agents provide the slowest result and the best sections. The present study on decalcification is a comparison of six different decalcifying agents and the optimum duration of their use, when used on a section of rat mandible and teeth. This study is a step forward in establishing the decalcification dynamics and identifying the method that combines the highest quality of stained sections with the shortest time.
The present study on decalcification was undertaken with the following aims and objectives:
- To study and compare the time taken for complete decalcification of the specimen by six different chemical agents.
- To study and compare the effect of various decalcification methods on cellular and nuclear changes of hard and soft tissues.
- To study and compare the effect of various decalcification methods used on the staining intensity with Ehrlich's Hematoxylin and Eosin stain.
- To compare all of the above and to determine the ideal decalcification technique.
| Materials and Methods|| |
The study was designed as an animal study. Evaluation of the time required for demineralization on a fixed sample size requires that the tissue samples be free of previous pathological mineral loss. Examination of the quality of preservation of soft tissue and cellular components after demineralization required the use of fresh samples to avoid inclusion of inadequately fixed and degraded tissue.Obtaining fresh samples from living donors would be unethical and, obtaining fresh samples from cadavers is not possible. Thus, it was essential to use rats in this study in order to obtain fresh specimens of teeth along with alveolar bone. Study samples were obtained from mandibles of rats cared for in the animal house. All protocols were followed as mentioned by the ethical committee of the college. The rats were sacrificed under chloroform anesthesia. Posterior mandibular segments with three molar teeth were used as specimens, thus providing us with two samples per rat. Posterior mandibular segments of rat were selected for the procedure since the crystal morphology and decalcification patterns of rat and human teeth are proved to be similar.  Six demineralizing agents [Table 1] were tested on a total of eight samples each. Twenty four rats were used, making the total size forty eight, which were sample coded as shown in [Table 2].
Formic acid and formal-nitric acid were chosen since they are the most common weak and strong demineralizing acids used respectively. Ethylene Di-Amine Tetra Acetic Acid (EDTA) was chosen since it is the mildest decalcifying agent according to the literature. All the specimens were weighed before the fixation. Specimens decalcified using EDTA and Perenyi's fluid were fixed in 10% formalin, since the EDTA formulation used did not contain any fixative and the ethyl alcohol contained in Perenyi's fluid was considered to be a slow fixative. The samples which were decalcified using formal nitric acid 8%, formal nitric acid 10%, formic acid 8%, and formic acid 10% were taken directly for the decalcification process as they had fixatives in them.
After the samples were properly labeled each was suspended in a coplin jar in approximately 100 ml of decalcifying agent, using a thread. The exact time at the start of decalcification was noted. The pH and temperature of the solutions was recorded on a daily basis. The end point of decalcification for both acids and EDTA was estimated using chemical test. A modification of this method by Crawford allowed the use of the chemical test following decalcification in Perenyi's fluid. 
Post decalcification, the samples were washed in running tap water for two to three minutes and then cut into three bits, each containing one molar. Then the tissues were subjected to normal processing in automated tissue processor using the same program for all the samples.The paraffin infiltrated tissues were embedded in wax blocks and were sectioned to a thickness of 5μm using semiautomatic soft tissue microtome (Leica RM 2165, Germany).
The sections were stained by Hematoxylin and Eosin (H and E) using Ehrlich's Hematoxylin which is an intense stain, especially useful for demonstrating the structure of decalcified sections. It was used regressively (Staining time 20-30 minutes). , The stained sections were examined using a light microscope.
The efficacy of different decalcifying agents used in the study was evaluated on the basis of following parameters:
- Time taken for decalcification based on chemical estimation of end-point.
- Effect on processing was assessed based on ribbon formation, scoring/splitting of sections during cutting and the ease with which the sections could be handled.
- Effect on staining was assessed by intensity of Hematoxylin staining of the nuclei and intensity of Eosin staining of the cytoplasm.They were assessed and graded as adequate or over stained or under stained. The stained sections were checked for the presence of cutting and reprecipitation artifacts. ,,
- Effects on histological detail of tissues: Histological sections are affected by many variables e.g., Fixation, processing, cutting technique, staining time etc.  All the above variables were kept consistent by the use of standardized techniques and recommended parameters.
- Dental pulp: The dental pulp was examined for the presence of all the four zones of the pulp and the amount of separation of pulp from the surrounding dentin.
- Dentin: The harmful effects of the decalcifying solutions on dentin were assessed based on the presence of vapor bubbles, fraying in the dentinal tubules and destruction of the odontoblast architecture.
- Cementum: The loss of Cementum architecture and loss of attachment from surrounding dentin or Periodontal Ligament (PDL) were assessed.
- Periodontal ligament: The PDL was examined for the amount of separation from surrounding hard tissues namely tooth and bone.
- Alveolar Bone: The Osteoblasts lining the trabeculae and Osteocyte retraction within the lacunae was checked and was rated as <50% of cells showing retraction and >50% of cells showing the retraction.
| Results|| |
Results were calculated as Mean ± SD or number and percentage. One way ANOVA was used for multiple group comparisons and Chi-square test was used for analyzing categorical data. P value of 0.05/less was set for statistical significance.
The decalcification was achieved within 1.7 days (40 hrs 48 min) by 10% formal nitric acid which was the fastest; whereas EDTA took the longest that is 17.9 days (429 hrs 36 min). 8% Formal nitric acid took 2.3 days (55 hrs 20 min), Perenyi's fluid took 2.5 days (60 hrs), whereas Formic acid 10% and Formic acid 8% took 16.3 days (391 hrs 20 min) and14.8 days (355 hrs 20 minutes) respectively. The difference was statistically significant [Table 3].
Observations showed that EDTA, 10% and 8% formal nitric acid provided samples which were sectioned without any tear, did not pose any difficulty in handling and which were not friable. Samples decalcified by 10 and 8% formic acid were more friable and difficult to handle. Perenyi's fluid was the worst with almost 90% of the tissue, posing difficulty in sectioning. The tissues were extremely friable and were difficult to handle. [Table 4] shows that the differences between the groups were statistically significant.
Specimens decalcified with EDTA showed the highest number (95%) of H and E sections without any over or under staining followed by formic acid 8% (62%) and formic acid 10% (54%). Formal nitric acid 10 and 8% gave the next best results with 45 and 41% of sections showing adequate staining respectively. The maximum number of over and under stained sections was shown by specimens decalcified with Perenyi's fluid. The difference was statistically significant [Table 5]a and b.
The osteoblasts lining was present in all the sections from the samples decalcified in all the solutions except for formal nitric 10% and formic acid 8%, which showed the presence of lining in 95.5 and 83% of cases respectively [Table 6].
Osteocyte retraction within the lacunae was checked and was rated as <50% of cells showing retraction and >50% of cells showing the retraction. The osteocyte retraction was remarkably less in case of formic acid 8% and EDTA when compared to other solutions, which showed osteocyte retraction, of more than 50%. This was statistically significant [Table 7].
[Table 8] shows that EDTA did not have any destructive effect on the cementum. Samples treated with Perenyi's fluid showed maximum destruction (86%). Formic acid 8% and formal nitric 8% showed fairly less destruction (54 and 50% respectively) followed by Formic acid 10% and formal nitric 10% (27 and 22% respectively). The difference in the results between the groups was statistically significant.
PDL separation from surrounding dentin was observed and was graded as mild, moderate and severe depending on the degree of separation of the fibres from surrounding dentin and bone. The highest number of sections (65%) showing mild separation of PDL from surrounding hard tissues was seen in samples treated with EDTA. Samples treated with Formic acid 10% showed maximum destruction with only 18% of cases with mild separation of PDL. Formal nitric 10%, formal nitric 8% and Formic acid 8% showed fairly less destruction with 36, 45 and 33% of cases with mild separation of PDL followed by Perenyi's fluid (27%). The difference in the results between the groups was statistically significant [Table 9].
Osteoblasts and the dentinal tubules were checked for the damage caused during decalcification techniques. [Table 10] shows that the dentin destruction was minimal (5%) in the samples treated with EDTA and maximum in the samples treated with Perenyi's fluid (86%). Formic acid 8% and formal nitric 8% showed fairly less destruction (70, 62% respectively) followed by Formic acid 10% and formal nitric 10% (40%). The difference in the results between the groups was statistically significant.
Pulp separation from surrounding dentin was observed and was graded as mild, moderate and severe depending on the degree of separation. A particularly interesting finding is the preservation of all the layers of pulp zones in all the samples of each group, irrespective of the quality of the section and the cellular details [Table 11].
The overall tissue integrity as seen under the microscope was well preserved in samples treated with EDTA, and they fulfilled all the histological criteria fairly well. Cellular structures could be well appreciated in all sections of EDTA, whereas they could not be seen in nearly half of the sections of Perenyi's fluid. Formal nitric of both the percentages10% and 8% gave fairly good cellular detail and were rapid in their action; formic acid 10% and 8% was the intermediate. The above findings were statistically significant.
| Discussion|| |
The choice of decalcifying agent used is largely dictated by the urgency of the procedure. Distortion and macerations are demonstrated by the use of chemicals. At a macroscopic level the tissue may appear unchanged, while microscopic evaluation reveals swelling, shrinkage, vacuolization, disruption, and fraying; not attributable to pathological conditions. 
The decalcification was achieved within 1.7 days (40 hrs 48 min) by 10% formal nitric acid which was the fastest; whereas EDTA took the longest that is 17.9 days (429 hrs 36 min). 8% Formal nitric acid took 2.3 days (55hrs 20 min), Perenyi's fluid took 2.5 days (60 hrs), whereas Formic acid 10% and Formic acid 8% took 16.3 days (391 hrs 20 min) and 14.8 days (355 hrs 20 minutes) respectively. The present study was compatible with the other studies by Geoffrey Brown and Bancroft which showed Formal nitric acid (with formaldehyde fixative) to be the fastest decalcifying agent taking 2-3 days' time to decalcify 5 mm thick bone.  The time taken for decalcification in the present study using EDTA was 17.9 days (429 hrs 36 min). This is because of the inherent mechanism of action of EDTA which acts purely as a chelating agent and hence takes longer to decalcify. But this was considerably less compared to the study by Preece, Culling, and White according to which EDTA took 6-12 weeks.  This was possible because the decalcifying solution of EDTA was modified as per the study of MooreRJ.
EDTA which was the slowest agent of all, resulted in samples most of which (70%) were sectioned very easily, were easy to handle and not friable. The fastest decalcifying agents 10% formal nitric acid and 8% formal nitric acid provided samples (59 and 54% respectively) that were readily sectioned, were easy to handle and produced sections most of which were intact and not shattered. Both findings were in agreement with a previous study which showed that samples decalcified by Formal Nitric acid resulted in better sections.  Samples decalcified by 8% formic acid were also sectioned easily (41.7%) but were also more friable, posing a difficulty in their handling. This was followed by formic acid 10% (13.6%). But the worst case was that of Perenyi's fluid in which most tissues (90%) posed severe difficulty in sectioning. The tissues were extremely friable and were difficult to handle which was contradictory to the findings of Geoffrey Brown.  Other agents produced intermediate results. The poor sectioning in case of Perenyi's fluid could be attributed to the strong effect of nitric and chromic acid on the tissue friability.
Specimens decalcified with EDTA showed the highest number (95%) of H and E sections with adequate staining (not over or under stained) followed by formic acid 8% (62%) and formic acid 10% (54%). Formal nitric acid 10 and 8% gave the next best results with 45 and 41% of sections showing adequate staining respectively. The maximum number of over and under stained sections was shown by specimens decalcified with Perenyi's fluid [Figure 1] and [Figure 2].The most pronounced effect of acid decalcification is the impairment of staining properties. This is dependent on the solution acidity and the time it will take to decalcify. ,, Thus, the quicker the decalcification, the greater will be the injury and its effects on H/E.  Following acid exposure nucleus stains poorly with cationic dyes such as Hematoxylin and cytoplasm over-stains by the briefest exposure to anionic dyes such as Eosin. This effect can be of diagnostic significance if such under stained nuclei are falsely interpreted as being non-viable. For this reason, staining procedures must be performed carefully following acid decalcification. , The acids used in the present study were in combination with fixative and since we had used Ehrlich's stain we could overcome the staining impairment.
Different histological criteria used for assessment of the sections were osteoblast lining, osteocyte retraction, pulp zones; pulp separation from dentin, dentin destruction, cementum destruction and PDL separation from the surrounding hard structures. e.g., The osteoblasts lining was present in all the sections [Figure 3] from the samples decalcified in all the solutions except for formal nitric 10% and formic acid 8%, which showed the presence of lining in 95.5 and 83% of cases respectively. The osteocyte retraction was remarkably less in case of EDTA when compared to other solutions, which showed osteocyte retraction, of more than 50% [Figure 4]. EDTA has little or no effect on tissues other than the bone mineral, which it removes by binding with calcium ions and gradually depleting the crystal from the outer layer.  On the other hand, Perenyi's fluid, which is a combination of twostrong acids - Nitric and Chromic acids, can be expected to cause extensive tissue damage.
|Figure 3: Osteoblast rimming the bonytrabeculae A ‑.Bony trabeculae B ‑.Osteoblastrimming|
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The separation of cementum from dentin and loss of tissue architecture was checked. The present study found that EDTA showed no cementum destruction and samples treated with Perenyi's fluid showed maximum destruction [(86%) [Figure 5] while Formic acid 8% and formal nitric 8% showed an intermediate level of destruction (54 and 50% respectively).This was followed by Formic acid 10% and formal nitric 10% (27 and 22% respectively). The cementum destruction can also be explained on the basis of lytic effects of the acids.
PDL separation from surrounding dentin was observed and was graded as mild, moderate and severe depending on the degree of separation of the fibers from surrounding dentin and bone. The highest number of sections (65%) showing mild separation of PDL from surrounding hard tissues was seen in samples treated with EDTA. Formal nitric 10%, formal nitric 8 and Formic acid 8% showed moderate destruction with 36, 45 and 33% of cases with mild separation of PDL followed by Perenyi's fluid (27%). Samples treated with Formic acid 10% showed maximum destruction [Figure 6] with only 18% of cases with mild separation of PDL. Pulp separation from dentinal border and preservation of cellular details is dependent on fixation, and the choice of decalcifying agent. Frequently obtaining a good histological result for the calcified tissues is not possible without some damage to the soft tissues.On the other hand, adequate preservation of the soft tissues leaves the specimen incompletely decalcified. Thus, it is difficult to fulfill the requirements for simultaneous analysis of mineralized and non-mineralized tissues. 
|Figure 6: PDL separation from surrounding hard tissue A ‑.Alveolar bone C ‑.Cementum D ‑.Dentin P ‑.Periodontal ligament|
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The destruction of odontoblasts, vapor bubbles and fraying in dentinal tubules in the present study, was negligible in case of EDTA. Formic acid 8% and formal nitric 8% showed fairly less destruction followed by Formic acid 10% and formal nitric 10% (40%).Greatest severity [Figure 7] was noted in case of Perenyi's fluid. The present study is in correlation with another study with similar results.The authors suggested that strong acid decalcification opened up the dentinal tubules quickly and served as a pathway to the pulp tissue, thus destroying or separating the pulp from dentin. This was the reason why fraying in the dentinal tubules along with any destruction of the odontoblast architecture was seen in sections obtained after using strong acids. 
A particularly intriguing finding is the presence of all the layers of pulp zones in all the samples of each agent, irrespective of the quality of the section and the cellular details. Pulp separation from dentin was least in EDTA group and 8% formal nitric acid. Pulp separation from surrounding was severe in other groups [Figure 8]; no satisfactory explanation could be found for this.
The overall difference in histological impression was statistically significant. The overall tissue integrity as seen under the microscope was well preserved in samples treated with EDTA. Cellular structures could be well appreciated in all the EDTA demineralized tissue sections, whereas they could not be seen in nearly half ofthe sections prepared by Perenyi's fluid demineralization. Formal nitric of both the percentages (10% and 8%) gave fairly good cellular detail and were rapid in their action, Formic acid 10% and 8% were the intermediate.
| Conclusion|| |
The final impression led to the proposition that EDTA was indeed the best decalcifying agent currently available and should be advocated where time factor is relatively un-important and for Immunohistochemistry. However, when the purpose of decalcification is the diagnosis and the surgeon is dependent on the pathologist for the biopsy report, a balance has to be struck. Since the present study has proved that formal nitric acid is not only a faster decalcifying agent, but also results in good quality sections with adequate staining, one can use formal nitric acid 10% or formal nitric acid 8% when time becomes a relevant factor.
| References|| |
|1.||Moore RJ. Bone. Available from: http://users.adam.com.au/royellis/BONE.htm. [Last accessed on 2012 Feb]. |
|2.||Simmelink JW, Abrigo SC. Crystal morphology and decalcification patterns compared in rat and human enamel and synthetic hydroxyapatite. Adv Dent Res 1989;3:241-8. |
|3.||Brown GG. A manual for the student, practicing technologist and resident in pathology. NewYork: Appleton-century-crofts; 1978. |
|4.||Callis GM, Bancroft JD. Theory and Practice of Histological Technique. 5 th ed. Edinburgh: Churchill Livingstone; 2004. |
|5.||Culling CF, Allison RT, Barr WT. Cellular pathology technique. 4 th ed. London: Butterworths; 1984. |
|6.||Morse A. Formic acid-sodium citrate decalcification and butyl alcohol dehydration of teeth and bones for sectioning in paraffin. J Dent Res 1945;24:143-53. |
|7.||Available from: http://users.adam.com.au/royellis/tp/tp.htm. [Last accessed on 2012 Feb 20 th ]. |
|8.||Crespi R, Grossi SG. A method for histological examination of undecalcified teeth. Biotech Histochem 1992;67:202-6. |
|9.||Fernandes MI, Gaio EJ, Rosing CK, Oppermann RV, Rado PV. Microscopic qualitative evaluation of fixation time and decalcification media in rat maxillary periodontium. Braz Oral Res 2007;21:134-9. |
|10.||Selvig KA. Ultrastructural changes in human dentine exposed to a weak acid. Arch Oral Biol 1968;13:719-26. |
Department of Oral Pathology, College of Dentistry, Gulf Medical University, Ajman
United Arab Emirates
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11]