Comparative histologic evaluation of teeth decalcified by conventional method versus microwave induced decalcification

Abstract

Introduction:

Complete demineralization of hard tissues with preservation of organic components to retain their tissue architecture and to obtain thin section for histological examination is done by a process known as “decalcification”. Tooth decalcification may be achieved by subjecting the tissue to a decalcifying chemical agent and employing heat, vacuum, or electric current to enhance the procedure. Completion of decalcification is adjudged by determination of end-point. Histological examination of hard tooth tissues and pulp is essential for diagnosis of developmental pathologies, pulp pathologies, and forensic odontology analysis as well as for research purpose.

Aim:

In our study, we studied the time consumed for decalcification procedure by conventional decalcification method and with the microwave-assisted decalcification method for teeth, using 5% nitric acid, 5% trichloracetic acid, and 14% EDTA (Ethylenediaminetetraacetic acid), respectively, along with morphological preservation of dentin and pulp tissue by analyzing preservation of their issue structure (pulp shrinkage, odontoblastic layer damage) and staining efficacy (patchy staining) for the three types of decalcifying agents used.

Materials and Method:

The sample for this study included 120 freshly extracted teeth consisting of teeth of mandibular dentition. Samples were divided into two study groups:

Group I: Decalcified by Conventional method

Group II: Decalcified by Microwave method.

Each group consisted of, three sub-groups employing a different decalcifying agent from among the three decalcifying agents used, that is, 5% nitric acids, 5% trichloroacetic acid, and 14%EDTA respectively.

Result:

The difference in speed of decalcification for both groups showed significant result. In either group, decalcification was fastest using 5% nitric acid. The difference in number of decalcified teeth sections showing shrinkage of pulp was not statistically significant for both groups. Damaged odontoblastic layer was evident only in the teeth treated with 5% nitric acid by microwave method. Patchy staining was observed in a few samples of all sample groups except those decalcified with EDTA by conventional method but the results were not statistically different on intercomparison.

Discussion and Conclusion:

Our results are similar to those of previous studies and we found that microwave method is an option that can reduce the time of decalcification. Tooth tissue morphology also was noted to be well preserved with 5% trichloracetic acid and 14% EDTA in comparison to 5% Nitric acid.

INTRODUCTION

Biopsies of head and neck pathology exhibit complex structure consisting soft as well as hard tissue.[1] Microscopic examination is imperative for the study of morphology and pathology of given specimen. Soft tissue can be easily sectioned and examined contrary to hard tissue which requires complete demineralization to obtain thin section for histological examination by a process known as “decalcification”.[2]

Decalcification was defined by Cook and Ezra-Cohn (1962) as “any treatment, which destroys the inorganic phase of the bone, with the removal of essential element calcium in such a way, so as, to leave the organic portion sufficiently intact to be handled and sectioned by ordinary methods.”[3]

The decalcification procedure completely removes the calcium salts from the calcified hard tissues, but, there is preservation of organic components which helps to retain the tissue architecture. Teeth being the hardest of animal tissues, with a high inorganic content, are difficult to prepare for microscopic examination. Decalcified sections of dental hard tissues are necessary to evaluate oral pathologies involving them. Furthermore diverse soft tissue pathologies may contain hard tissue within them owing to certain dystrophic or metaplastic tissue responses and such soft tissue specimen may, therefore, require to be decalcificied for proper analysis and histopathological report.[4,5] Histological examination of hard tooth tissues and pulp is essential for diagnosis of developmental pathologies, pulp pathologies and forensic odontology analysis as well as for research purpose.[6]

Decalcification of hard tissues is essentially required to obtain thin sections of the hard tissues which is otherwise not possible by ordinary histological methods. Tooth decalcification may be achieved by subjecting the tissue to a decalcifying chemical agent and employing heat, vacuum or electric current to enhance the procedure. Decalcifying agents are the chemicals that are used for the process of decalcification. The decalcifying/chelating agents commonly used are either strong mineral acids, weaker organic acids or chelating agents.[5]

The process of decalcification usually takes few to many days and additionally it also reduces the quality of the tissue staining, therefore attempts are still ongoing to find an optimal method for accelerating the decalcification procedure. For this purpose, increasing the temperature of tissue specimen in permissible limits, subjecting tissue to vibrator, placing it in vacuum or employing electric current to generate undulations in the decalcifying solution are some of the methods used. (Verdenius and Alma1958). Nowadays, using microwave oven for rotating and heating the tissue simultaneously (Sangeetha et al. 2013) as well as use of ultrasonic apparatus (Hatta et al. 2014) is also done to reduce the time taken for decalcification.[6,7,8,9]

As an alternative approach to rapidifying decalcification in tissue processing currently, tissue immersed in decalcifying agent is subjected to the rotation and heat in a microwave oven for repeated short spans at regular interval and the decalcifying solution is also changed at definite periodical intervals until the achievement of end point. It has already been demonstrated that when bone is decalcified by this method, it leads to a reduction of decalcification time and so the decalcification time decreases to 1/10^th of the time taken in the conventional method.[10]

Completion of decalcification is adjudged in many ways. The methods commonly used include, physical, chemical, or radiological methods. Other methods are also employed but, they need accurate mathematical quantification of residual calcium in the tissue by colorimetric method, flame photometric method, and spectrophotometric technique.[6]

In our research project, we had sought to analogize the time consumed for decalcification procedure by conventional decalcification method and with the microwave-assisted decalcification method for teeth, using 5% Nitric acid, 5%Trichloracetic acid, and 14% EDTA (Ethylenediaminetetraacetic acid) respectively, and thereby we attempted to find an ideal decalcifying agent in terms of the method of decalcification with corresponding influencing factors like speed of decalcification, preservation of tooth tissue structure (pulp shrinkage, odontoblastic layer damage) and staining efficacy (patchy staining) for the three types of decalcifying agents used.

MATERIALS AND METHODS

The sample for this study included 120 freshly extracted teeth consisting of teeth of mandibular dentition from the Department of Oral and Maxillofacial Surgery, GDC Raipur. Samples were divided into 2 study groups:

Group I: Decalcified by Conventional method

Group II: Decalcified by Microwave method.

Each group consisted of, three sub-groups employing a different decalcifying agent from among the three decalcifying agents used that is, 5% nitric acids, 5% trichloroacetic acid, and 14%EDTA respectively with, 20 samples in each sub-group. Both method groups used 60 teeth samples each with same number of each type of mandibular teeth. The specimen were fixed in 10% buffered formalin. Nitric acid is a strong inorganic acid which can only be used in a 5% dilute form for near complete decalcification of highly mineralized tissues like teeth. In greater concentration it will damage soft-tissue structure and disrupt cellular integrity whereas in lower concentrations it may fail to penetrate the tissue in its entire thickness. Similarly, Trichloracetic acid is a weak organic acid and so complete decalcification of highly calcified body tissue like tooth with it requires 5% conc. of acid for the process to be achieved in about 10 days. Higher conc. of this acid also damages tissue and cellular architecture and use of lower concentration does not ensure sufficient decalcification. Chelating agent like EDTA, on the other hand, is required to be used at a higher conc. of 14% for complete decalcification of teeth because, it primarily acts by binding to metallic ions like calcium and magnesium. However, even when it preserves tissue and cellular architecture better, it is a much slower decalcifying agent as compared to acids.

Inclusion criteria

Freshly extracted sound teeth which were extracted for orthodontic purpose or because they had grade III mobility or impacted teeth were collected.

Exclusion criteria

1. Teeth with caries.

2. Teeth with restoration.

3. Teeth associated with any pathology.

All the specimens were weighed before the study for standardization of procedure. After the samples were properly labeled, each sample was put in a beaker in 100 ml of decalcifying agent for decalcification. The exact time at the start of decalcification was noted. The decalcifying solutions were changed, pH and temperature was noted daily.

In the conventional method, we washed the formalin fixed tooth in running water for about half an hour prior to placing it in the decalcifying solution that was at normal temperature. The decalcifying solution was changed daily irrespective of the type of the solution until the end point was reached. (Clayden 1952).[7]

For microwave technique, a domestic microwave oven (LG Intellowave, Model 1911HE) which had a fixed rotary plate and a maximum power output of 700 W and input voltage 230 V-50 HZ with 41-43° C temperature was used. In this technique, the tooth suspended in 5% nitric acid solution as well as the tooth immersed in 5% trichloracetic acid were microwaved for 8 seconds each with 1-h interval between each microwave cycle for six times in a day. For 14% EDTA, the tooth immersed in it was microwaved for hourly cycles of 10 seconds each six times per day. The EDTA cycle was longer by two seconds as it is a chelating agent and so, has a slower decalcifying/chelating onset as compared to the acids used. During this tenure the specimen was not removed from microwave, so that, the temperature of all three decalcifying solutions that were microwaved was sustained in the range of 41-43°C. The decalcifying acid and/or EDTA solution was changed every day until the end-point of decalcification was reached. The decalcifying agent used and changed was checked everyday to note if, the end-point was achieved. End-point was determined by chemical method using ammonium oxalate (Clayden 1952).[7]

End-point was determined by chemical method using Ammonium oxalate for both conventional and microwave method. Five ml of decalcifying fluid needs to be neutralized with N2.NaOH prior to end-point evaluation. Then, 1 ml of 5 per cent ammonium oxalate was added. Turbidity of the fluid was the indicator of the presence of calcium. Absence of turbidity after a delay of 5 minutes indicated that the decalcifying fluid is free of calcium, hence decalcification was considered complete. It should be kept in mind that, testing for decalcification by the flexibility of the tissue is unreliable, and the out-dated practice of inserting a needle to feel for calcium deposits causes damage to the tissue.[5]

After decalcification was complete all tooth specimen were subject to routine processing of specimen followed by sectioning and H&E staining of tooth sections of both the groups. The stained decalcified teeth sections were compared for speed of decalcification, as well as preservation of tissue morphology and architecture besides staining efficacy for both groups as well as their sub-groups using the criteria proposed by Sangeetha et al.[7] between two groups as well as their sub-groups by two observers individually without providing data of type of decalcifying agent used or information of method used. The observations were then summarized and analyzed using Chi-square tests. The approval from the ethics committee is obtained and the date of approval is 25^th Nov 2022.

RESULTS

Complete decalcification of the extracted teeth using conventional method for 5% nitric acid, 5% Trichloroacetic acid, and 14% EDTA was achieved in 5, 3, and 91 days, respectively whereas for the microwave method the time taken was 3, 4, 33 days, respectively [Table 1]. Thus, the difference in speed of decalcification for both groups showed significant result. In either group, decalcification was fastest using 5%Nitric acid.

Table 1.

Time duration for conventional and microwave decalcification in days for the tooth specimens

	Method	N	Mean	Std. deviation	P
5% Nitric acid	Conventional method	20	4.7000	0.57124	0.001
	Microwave Induced method	20	2.7500	0.44426
5% Trichloracetic acid	Conventional method	20	4.4500	1.05006	0.003
	Microwave Induced method	20	3.5500	0.68633
14% EDTA	Conventional method	20	91.1500	5.13271	0.001
	Microwave Induced method	20	33.4000	4.93537

H&E stained slides of the decalcified teeth sections were evaluated to assess the preservation of tissue tissue morphology and architecture (shrinkage of pulp, damage to odontoblastic layer) and staining characteristics (patchy/uniform). Shrinkage of pulp away from dentinal wall was observed in 90% specimen for 5% nitric acid, in 80% specimen for 5% trichloroacetic acid and in 90% specimen for 14% EDTA in teeth sections decalcified by conventional method. For teeth sections of teeth decalcified by the microwave method, shrinkage of pulp was found to be in 100%, 90%, and 100% specimen decalcified in 5% nitric acid, 5% trichloroacetic acid and 14% EDTA, respectively [Graph 1, Figure 1a and b]. The difference in number of decalcified teeth sections showing shrinkage of pulp was not statistically significant for both groups

Graph 1.

Distribution of pulp shrinkage from dentinal tubules in tooth sections

Figure 1.

(a and b) Pulp shrinkage from dentinal wall (c and d) Damaged odontoblastic layer (e and f) Patchy staining

The odontoblastic layer was found to be damaged in 15% the specimen samples treated with 5% nitric acid by microwave method but, no such damage was seen in teeth decalcified by conventional method for the same acid. On the other hand, no damage to the odontoblastic layer was identified in the samples treated with 5% trichloroacetic acid and 14% EDTA irrespective of the method used. [Graph 2, Figure 1c and d]

Graph 2.

Distribution of damaged odontoblastic layer in tooth specimen

Patchy staining pattern was seen in 25% of specimen samples decalcified by conventional method compared to only 10% of samples decalcified by microwave method when 5% nitric acid was used. Patchy staining was evident in 15% specimen samples decalcified by conventional method using 5% trichloroacetic acid or 14% EDTA. However, patchy staining was not observed in specimen sections decalcified by microwave method with both 5% trichloroacetic acid and 14% EDTA. These results on comparison between two groups were not statistically significant. [Graph 3, Figure 1e and f]

Graph 3.

Distribution of patchy staining in tooth specimen

Additionally, yellow discoloration of the teeth sections was observed in specimen decalcified with 5% nitric acid but it was absent in specimen decalcified with 5% trichloroacetic acid and 14% EDTA irrespective of the method used.

DISCUSSION

Tooth decalcification is very easily influenced by the technique as well as decalcifying reagent used. At the same time, it is a procedure required to be routinely done among the laboratory procedures of oral pathology, in order to study the tooth structure, pulp calcifications, and also to evaluate the biological response of dental pulp to restorative materials and physical or chemical insults. A plethora of efforts are being put in to find a novel decalcifying agent or to modify the known decalcifying agents[7] in order to achieve optimum decalcification in a reasonable amount of time and with maximum preservation of the tissue architecture and morphology.[5]

The biggest pitfall is the time taken in the decalcification of a tooth which may span through a few weeks. Another important concern of decalcifying teeth is the extent of preservation tissue architecture and morphology that in turn depends on the decalcifying agent used and quality of the process of decalcification. Among many methods used so far, method using microwave oven has been seen to increase the speed of decalcification process.

In this study, we have assessed the speed of decalcification using two different methods for three types decalcifying agents and then compared the results for each group with respect to time taken for decalcification and comparison of quality of decalcification was done by noting various microscopic parameters of the decalcified tooth sections for both conventional and microwave method.

The decalcified tooth sections of teeth which were decalcified in nitric acid solution, a yellow pigmentation was observed irrespective of the method used. However, sections of teeth decalcified in other decalcifying agents did not show any discoloration of the teeth. This observation was in accordance with the study done by Sangeetha et al.[7]

Pulp tissue shrinkage away from dentinal wall was observed in decalcified teeth sections decalcified with 5% nitric acid irrespective of the method used whereas it was not evident in decalcified teeth sections decalcified in 5% trichloroacetic acid and 14% EDTA irrespective of the method used. The difference in pulp tissue shrinkage of decalcified teeth sections decalcified in 5% tricloroacetic acid and 14% EDTA was, statistically insignificant. Also, both 5% tricloroacetic acid and 14% EDTA had shown the good preservation of tissue architecture and morphology with both the methods. We, therefore, concluded that shrinkage of pulp away from dentinal wall in decalcified teeth sections may be affected by fixation and processing techniques also and not only by the decalcifying agent used.[7]

Similarly, microscopic observation of damage to odontoblasts may also influenced by the type of fixative and fixation method used besides the choice of decalcifying agent.[10]

Majority of decalcified tooth sections of teeth decalcified in nitric acid by conventional method showed microscopic evidence of discontinuous or lost odontoblastic layer and a few cases of the same change were also seen for sections of teeth decalcified with nitric acid but with microwave method. 5% tricloroacetic acid and 14% EDTA have shown no damage with both the methods.[11,12,13,14,15]

In present study, good cellular and nuclear staining was present in all the tooth sections decalcified with conventional as well as microwave-assisted method irrespective of the decalcifying agent used.

From our results of this study we interpreted that, the time taken for decalcification is significantly decreased using microwave technique regardless of the decalcifying agent used. This observation of our study, was in accordance with that of the study done by Hajioseini et al., Vongsavan et al., and Pitol et al. in which also they had observed a 30-fold increase in decalcifying speed with microwave technique.[12,13,14]

Patchy staining was evident in the entire sample decalcified with conventional method irrespective of decalcifying agent used. Few samples showed patchy staining with 5% nitric acid with microwave technique. None of the samples showed patchy staining with microwave method with 5% tricloroacetic acid and 14% EDTA. Patchy or inconsistent staining may result from very short application time of staining. It can be reduced by, doing the decalcification end-point test, post-decalcification acid removal, and time adjustment in staining procedure. Despite the fact that teeth were decalcified by either conventional or microwave method, it was observed in our study that, there was no statistically significant difference in the preservation of pulp tissue architecture and morphology of tooth tissues for any of the three decalcifying agents that were used in our study. However, the uniformity of decalcification was more in the microwave method in our study, as compared to conventional method, probably owing to the agitation that the teeth were subjected to during microwave decalcification thereby facilitating better penetration of decalcifying agent.[11,12,13,14,15,16]

From the results obtained in our study we draw the inference that, 5% trichloroacetic acid and 14% EDTA showed better results as compared to 5% nitric acid in both the groups for all the parameters analyzed in our study. As the process of decalcification is slow with 14% EDTA irrespective of method used hence, in those cases in which there is urgency of histopathological analysis of the decalcified tissue, 5% trichloroacetic acid would prove to be a better alternative for hard tissue decalcification by either of the methods. Thus, both acids as well as chelating agent have their own merits and limitations.

CONCLUSION

The limitation of our study is that although we could find a decalcifying agent which is both time efficient and gives qualitatively reasonable decalcified teeth sections we could not evaluate its efficacy for all teeth of dentition. However, it was also observed that, the decalcification was faster as well as of better quality when done with microwave technique. In future, studies with a larger sample size and also with an inter-comparison of a greater number of decalcifying agents might give us, a more efficient decalcifying agent that is less time consuming coupled with an ability in it of better preservation of tissue architecture and so lesser deleterious effects on the tissue structure.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.