Optimization of Formic Acid-Formalin-Based Decalcification Protocol for Rat Calvarial Bone Histology

S. Amitha Banu; Khan Sharun; Merlin Mamachan; Athira Subash; Vadapalli Deekshita; Kirtika Sharma; Karikalan Mathesh; Obli Rajendran Vinodh kumar; Swapan Kumar Maiti; Abhijit M. Pawde; Laith Abualigah; Kuldeep Dhama; Amarpal

doi:10.18006/2024.12(2).218.225

Authors

S. Amitha Banu Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-7182-8169
Khan Sharun Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0003-1040-3746
Merlin Mamachan Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0009-0002-5861-1878
Athira Subash Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0009-0007-0947-704X
Vadapalli Deekshita Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0009-0005-8297-3475
Kirtika Sharma Centre for Wildlife, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0009-0003-2578-5828
Karikalan Mathesh Centre for Wildlife, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-2013-9179
Obli Rajendran Vinodh kumar Division of Epidemiology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-7232-4122
Swapan Kumar Maiti Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-2593-012X
Abhijit M. Pawde Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0001-7399-5596
Laith Abualigah Artificial Intelligence and Sensing Technologies (AIST) Research Center, University of Tabuk, Tabuk 71491, Saudi Arabia https://orcid.org/0000-0002-2203-4549
Kuldeep Dhama Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0001-7469-4752
Amarpal Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India https://orcid.org/0000-0002-4827-3433

DOI:

https://doi.org/10.18006/2024.12(2).218.225

Keywords:

Decalcification, Rat, Calvarial bone, Formic acid, Histopathology, Bone regeneration

Abstract

Decalcification is crucial in histological processing, particularly for studying mineralized tissues like bone. The choice of decalcification method can significantly impact the quality of histological sections and the preservation of tissue morphology. This study aims to establish a standardized protocol for decalcifying rat calvarial bone using a formic acid-formalin-based decalcification solution. The protocol was systematically optimized and evaluated based on various parameters, including decalcification time, formic acid concentration, and tissue integrity preservation. The decalcification process was evaluated through comprehensive assessments, including gross physical examination, chemical analysis, and radiographic imaging techniques. Our result demonstrated that the 10% formic acid concentration proved most effective for decalcifying rat calvarial bone samples within eight days, excelling in mineral content removal while preserving specimen structural integrity. In contrast, the 5% concentration failed to complete decalcification within ten days, and the 15% compromised sample quality within eight days. Histological analyses confirmed the efficacy of the 10% formic acid concentration in maintaining tissue integrity and achieving optimal staining quality. The standardized protocol presented in this study provides an effective and reliable approach for achieving consistent and high-quality histological sections of rat calvarial bone. An ideal decalcification agent should effectively remove calcium salts, preserve structural integrity and molecular components, facilitate rapid yet minimally damaging decalcification, and ensure ease of handling for laboratory personnel. Further exploration of its applicability to different bone types or species is recommended to broaden its research utility.

Author Biographies

Abhijit M. Pawde, Division of Surgery, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India

Centre for Wildlife, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India

Laith Abualigah, Artificial Intelligence and Sensing Technologies (AIST) Research Center, University of Tabuk, Tabuk 71491, Saudi Arabia

Hourani Center for Applied Scientific Research, Al-Ahliyya Amman University, Amman 19328, Jordan

Computer Science Department, Al al-Bayt University, Mafraq 25113, Jordan

MEU Research Unit, Middle East University, Amman 11831, Jordan

Applied Science Research Center, Applied Science Private University, Amman 11931, Jordan

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