Histological Evaluation of the Effectiveness of Four Decalcifying Solutions on Teeth and Bone in Rats: An In-vitro Study

Abstract

Introduction: Intense histological research has been conducted on human bone and teeth for a long period of time. Histological evaluation of these highly mineralised tissues demands a thorough decalcification process using different chemical agents, which might alter some aspects of tissue architecture and staining properties. Aim: To assess the impact of four decalcifying agents on rat teeth and bone, focusing on the rate of decalcification, staining effectiveness, and tissue structure preservation. Materials and Methods: An in-vitro animal study was conducted in the Department of Oral Diagnosis at the College of Dentistry, University of Baghdad, Bab Almoaadim, Baghdad, Iraq from October 2022 to March 2023. Four decalcification agents, namely 10% Neutral Ethylenediaminetetraacetic Acid (EDTA), 10% formic acid, 3% nitric acid, and 5% nitric acid, were used to decalcify 24 molar teeth with their surrounding alveolar bone obtained from healthy male rats, which were randomly divided into four groups, each group consisting of six teeth. The decalcified sections underwent regular processing, and staining was performed using Haematoxylin and Eosin (H&E). Grading was conducted after two separate observers examined the stained sections under a light microscope. Data were presented using Mean±Standard Deviation (SD), number, and percentage. A one-way Analysis of Variance (ANOVA) (F-test) was used to compare the decalcification times within groups. Several group comparisons, including categorical data, were examined using the Chi-square test. Results: Nitric acids (3% and 5%) exhibited the fastest decalcification with a mean value of (2±0.89) days, while Formic acid required (3.5±0.54) days, and EDTA was the slowest, with (19±1.09) days. These differences in decalcification time were found to be statistically significant (p<0.00001). Formic acid 10% demonstrated superior tissue preservation and staining quality, with excellent staining results, minimal dentin-pulp separation, and preservation of pulp zones, cementum, and osteoblasts. In contrast, nitric acid 5% resulted in severe dentin-pulp separation, absence of pulp zones, absence of osteoblast, and significant osteocyte retraction. Statistical significance was observed across all agents for dentin-pulp separation, pulp organisation, cementum destruction, Periodontal Ligament (PDL) separation, and osteocyte retraction (p-values of 0.004, 0.02, 0.02, 0.04, and 0.03, respectively). Conclusion: Consequently, 10% formic acid emerged as the most efficient decalcifying solution, ensuring rapid decalcification with favourable staining intensity and tissue architecture.

Keywords

• formic acid
• neutral ethylenediaminetetraacetic acid
• nitric acid
• tissue architecture