Comparison of Different Decalcification Methods Using Rat Mandibles as a Model

Abstract

Selection of decalcification agents is an essential consideration when processing mineralized tissues because the integrity and immunohistochemical characteristics of the tissues may be affected. Here, we report results obtained from the decalcification of rat mandibles using 10% ethylenediaminetetraacetic acid (EDTA) at room temperature (RT), 10% EDTA at 37C, 5% nitric acid, and 10% formic acid at RT. Decalcification endpoints were determined by microcomputed tomography. Morphological preservation and antigenicity were evaluated by hematoxylin and eosin staining and immunohistochemistry. Decalcification of the anterior and posterior portions of the mandible took 220 and 191 hr in 10% EDTA RT, 102 and 73 hr in 10% EDTA 37C, 13.5 and 4.3 hr in 5% nitric acid, and 140 and 36 hr in 10% formic acid, respectively. Decalcification in 10% EDTA at 37C was accelerated, but 10% EDTA at RT provided optimal results for immunohistochemistry and cellular and structural details. Decalcification using 5% nitric acid was accomplished in the shortest time and exhibited good cellular and architectural morphology, whereas 10% formic acid was suboptimal with respect to tissue and cellular morphology. Despite being the slowest method, EDTA at RT is still the recommended method for decalcifying mineralized tissues; however, if rapid decalcification is needed, 5% nitric acid is the best option, yielding acceptable tissue integrity and speed.

Investigation of Sustained BMP Delivery in the Prevention of Medication‐Related Osteonecrosis of the Jaw (MRONJ) in a Rat Model

“…Biosci. 2019, 19,1900226 starPEG-RGD-heparin hydrogels and polypropylene fumarate hydrogels. [25] StarPEG-RGD-heparin based hydrogels are particularly advantageous, as they can be functionalized to mimic the extracellular matrix.…”

“…Decalcification of samples for paraffin embedding was carried out in Ethylenediamine tetraacetic acid (EDTA) at 37 °C (Kos Milestone microwave, ABACUS, Brisbane, Australia). Solutions were changed weekly . After the decalcification was completed, the specimens were processed overnight in an Excelsior ES Tissue Processor (Thermo Scientific, Brisbane, Australia), prior to embedding in molten paraffin wax at 60 °C at a Thermo Shandon Histocentre 3 Embedding Station (Thermo Scientific, Brisbane, Australia).…”

“…Biosci. 2019, 19,1900226 for analysis of all samples using resin and paraffin histology. Decalcification of samples for paraffin embedding was carried out in Ethylenediamine tetraacetic acid (EDTA) at 37 °C (Kos Milestone microwave, ABACUS, Brisbane, Australia).…”

“…Solutions were changed weekly. [19] After the decalcification was completed, the specimens were processed overnight in an Excelsior ES Tissue Processor (Thermo Scientific, Brisbane, Australia), prior to embedding in molten paraffin wax at 60 °C at a Thermo Shandon Histocentre 3 Embedding Station (Thermo Scientific, Brisbane, Australia). Sections were cut at 5 µm with a Leica RM2235 rotary microtome (Leica Biosystems, Nussloch Germany).…”

“…Biosci. 2019, 19,1900226 regions surrounding the osteoclasts were counted. Statistical analysis was carried out using a one-way ANOVA test to determine significance (p < 0.05).…”