Comparing the effects of three different decalcifying agent on extracted tooth-an in vitro study

Background: Head and neck is a complex structure of both soft and hard tissues, soft tissues put forth little resistance to the histochemical techniques. But hard tissues possess unique characteristic of hardness and sensitive procedures in the histopathology laboratory. The hard tissue may arrive at the laboratory for a number of reasons. Various pathologies may be associated with this hard tissue for which they need to be processed and stained so as to assist the pathologist in their reporting. For this reason the preservation of hard tissue close to the living state is essential for understanding of cellular and subcellular structures and functions. Before any calcified tissue can be processed and sectioned conventionally, the calcium must be removed. This process is called “decalcification” which is a chemical process that helps in further sectioning of the histological specimen. This can be done using chemical solutions like acids and chelating agents for preserving the organic skeleton.
Aims and objective: In the present study comparison between three different decalcifying agents with respect to rate of decalcification, the effect of decalcifying agents on the dental tissues, and its influence on the staining characteristics are being studied. Various methods for end point determination of decalcification can be done by chemical, physical, radiological and by weighing the specimen. The mechanical and radiological method does not allow the course of the procedure to be followed strictly. Thus, the need of quantitative analysis of calcium estimation from the decalcifying solution arose which was carried out using atomic absorption spectrophotometer.
Material and Methods: 45 freshly extracted human premolar teeth were taken within the age group of 15-25 years. They were then separated randomly into three groups of 15 each for decalcification in three different solutions.. The three groups include Group A-5% nitric acid, Group B-5% Formalin-Formic acid, Group C-5% EDTA. End point of decalcification of solutions were assessed using the chemical method. Decant decalcifying solutions that were saturated with calcium ions were thereafter sent for quantitative calcium estimation using atomic absorption spectrophotometer to confirm if end point of decalcification determined was accurate. The decalcified teeth were then routinely processed, sectioned, and stained with hematoxylin and eosin stains and observed under microscope by two independent observers and grading was done.
Results: Considering preservation and staining characteristics of both hard and soft tissues, superior results were obtained with 5% EDTA followed by 5% nitric acid and formic acid - formalin which was according to the respective mean values obtained.
Conclusion: EDTA, though being the slowest decalcifying agent among the three agents used in the study, gave excellent results for soft-tissue integrity, and best quality of both soft-tissue and hard-tissue staining.