Tissue integrity, costs and time associated with different agents for histological bone preparation

Abstract

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin-fixed and paraffin-embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morsés solution (Group V). Serial, 3-μm-thick sections were obtained and stained with hematoxylin-eosin to calculate the percentage of osteocyte-occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi-quantitative analysis to verify the adjacent muscle-to-bone integrity and preservation of collagen fibres. The highest percentage of osteocyte-occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8-5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p < .001). Group IV (EDTA) had the highest integrity of muscle and collagen type I and III (P < 0.01). Demineralization with 10% acetic acid was the most effective at preserving bone tissue, while 5% EDTA was the best at maintaining collagen and adjacent muscle to bone. In conclusion, nitric acid at 5% showed the most efficient result as it balanced both time and cost as a demineralizing solution.

Matrix mineralization controls gene expression in osteoblastic cells

“…Importantly, demineralization of the surface did not result in any detectable molecular changes to the osseous surface other than the depletion of mineral. With respect to EDTA, this observation is in line with reported data showing preservation of the organic portion of the matrix post EDTA treatment [21]. We believe that the molecular characterization of osseous surface models provides an important benchmark in the future development of studies addressing the interaction of bone cells with osseous surfaces.…”

Tissue Morphology and Antigenicity in Mouse and Rat Tibia: Comparing 12 Different Decalcification Conditions

“…47,48 Some studies have reported acceptable results obtained with hydrochloric or nitric acid. 10,22,26,44 In contrast, the findings from this study were in agreement with Neves et al, who found specific positive staining despite poor tissue morphology, and concluded that hydrochloric acid should be avoided for bone decalcification if preservation of bone matrix and antigenicity is desired. 49 Moreover, Athanasou et al reported that sufficient fixation and controlled decalcification in hydrochloric and nitric acids had no negative effects on diagnostic markers, such as vWF or prostate-specific antigen.…”

“…When decalcified in EDTA, formic acid, or nitric acid, the cartilage showed weaker staining at 37C. These findings complement the results of several other studies, confirming that both fixation and decalcification methods have been associated with proteoglycan and glycosaminoglycan depletion 4,26,27,32 reducing the staining intensity of Safranin O, which is directly proportional to the proteoglycan content in normal cartilage. 37 Optimal fixation and decalcification conditions for cartilage-containing specimens should always be decided in a pilot study.…”

“…19 Previously published studies comparing different decalcification methods for mineralized tissues mostly used teeth and mandibular bone of human and rat origin [20][21][22][23][24][25] or rat femurs. 10,26,27 Therefore, the main goal of this study was to evaluate and compare commonly used decalcification methods for mouse and rat tibiae. To investigate this, micro-computed tomography (microCT) was used to accurately measure rates of decalcification for exact demineralization end points and to determine the effects different decalcification solutions and temperatures have on the preservation of tissue morphology and antigenicity of mouse and rat bone tissue.…”

JDRHistorical Highlights #13

“…She shared her vast experience in her paper and gave detailed insights into the problems and troubleshooting methods she had developed over the years. “Morse’s solution” or “Anna Morse solution” is still widely used in hard tissue histology and performs well in modern histology laboratories as a reliable reagent for rapid and effective tissue decalcification (Abrantes et al 2017). Morse moved to the Harvard School of Dental Medicine, where she continued to develop histological methods, focusing on the visualization of enzyme activity in hard tissue specimens.…”