A comparative study of various decalcification

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Original Research A comparative study of various decalcification techniques Prathibha Prasad, Mandana Donoghue1 Department of Oral Pathology, College of Dentistry, Gulf Medical University, Ajman, UAE. 1 College of Dental Sciences, Davangere, Karnataka, India

ABSTRACT

Received : 13‑03‑12 Review completed : 16‑08‑12 Accepted : 25‑11‑12

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. Key words: 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

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.[1] Dental pulp evaluation is often a part of research protocols followed in assessment Address for correspondence: Dr. Prathibha Prasad E‑mail: [email protected] Access this article online Quick Response Code:

Website: www.ijdr.in PMID: *** DOI: 10.4103/0970-9290.117991

Indian Journal of Dental Research, 24(3), 2013

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. 302


A comparative study of various decalcification techniques

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.[2] 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. 303

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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.[3] 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 Table 1: The demineralizing agents used and their composition Formal nitric acid 10% 40% formaldehyde Concentrated nitric acid Distilled water[3] Formal nitric acid (8%) 40% Formaldehyde Concentrated nitric acid Full volume with distilled water[3] Formic acid 10% Formic acid (S.G. 1.20) 10% formal saline[3] Formic acid formalin (Gooding and Stewart (8%) 90% stock formic acid Formaldehyde (37‑40%) Distilled water[4] Perenyi’s fluid 10% nitric acid 0.5% chromic acid Absolute ethyl alcohol[3] Ethylene Di‑Amine Tetra Acetic Acid Solution A Sodium di‑hydrogen orthophosphate Distilled water Solution B Di‑sodium hydrogen orthophosphate (Anhydrous) Distilled water EDTA[1]

5 ml 10 ml 85 ml 4 ml 8 ml to 100 ml 10 ml 90 ml 80 ml 50 ml 870 ml 4 parts 3 parts 3 parts 31.2 g 1 liter 28.4 g 1 liter 140 g

Table 2: Sample coding Type of decalcifying agent Formal nitric acid (10%) Formic acid (10%) Formal nitric acid (8%) Formic acid (8%) Perenyi’s fluid EDTA

Sample coding A1, A2, A3, A4, A5, A6, A7, A8 B1, B2, B3, B4, B5, B6, B7, B8 C1, C2, C3, C4, C5, C6, C7, C8 D1, D2, D3, D4, D5, D6, D7, D8 E1, E2, E3, E4, E5, E6, E7, E8 F1, F2, F3, F4, F5, F6, F7, F8

Indian Journal of Dental Research, 24(3), 2013


A comparative study of various decalcification techniques

20‑30 minutes).[4,5] 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.[3‑5] • Effects on histological detail of tissues: Histological sections are affected by many variables e.g., Fixation, processing, cutting technique, staining time etc.[5] 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 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 Indian Journal of Dental Research, 24(3), 2013

Prasad and Donoghue

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 [Tables 5a and b]. Table 3: Number of days required for decalcification Days‑Mean 1.7 2.3 16.3 14.8 2.5 17.9

Formal nitric acid 10% Formal nitric acid 8% Formic acid 10% Formic acid 8% Perenyi’s fluid EDTA ANOVA F P

SD 0.3 0.4 1.3 0.7 0.4 2.0

441.8 >0.001, HS

EDTA=Ethylene Di-Amine Tetra Acetic Acid

Table 4: Ease of sectioning

Formal nitric acid 10% Formal nitric acid 8% Formic acid 10% Formic acid 8% Perenyi’s fluid EDTA

Easy Difficult Very difficult Total n (%) n (%) n (%) n 13 (59.1) 5 (22.7) 4 (18.2) 22 13 (54.2) 6 (25.0) 5 (20.8) 24 3 (13.6) 9 (40.9) 10 (45.5) 22 10 (41.7) 6 (25) 8 (33.3) 24 2 (9.1) 14 (63.6) 6 (27.3) 22 14 (70) 5 (25) 1 (5) 20

χ2=32.9, P