Decalcification Agents Research Articles

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

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 dentinpulp 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.

Evaluation of Different Decalcification Agents on Extracted Tooth: A Comparative Study.

The pulp is the most negatively impacted tissue during decalcification since it comprises the soft tissue components. The most effective decalcifying agent would be safest for cells and tissues while yet removing all traces of calcium. It has to get the job done quickly and have good staining properties. The goal of this research was to identify the most effective decalcifying agent for diagnostic purposes via a qualitative investigation of tissue preservation and a comparison of the efficiency of several decalcifying agents on human permanent teeth, covering both hard and soft tissue components. Fifty premolars from people aged 14 to 30 who needed them pulled for orthodontics were included in the research. Participants in the research were divided into five groups of ten. Group A, Group B, Group C, Group D, and Group E make up the total of five groups. In this investigation, we compared the efficiency of five decalcifying chemicals and analyzed their staining patterns and effects on tooth tissue. Fifty premolar teeth from participants aged 14-30 years old were removed for orthodontic therapy. For the research, they were split up into five groups of ten. Group A contains 5% ethylenediaminetetraacetic acid (EDTA), Group B contains 10% formic acid, Group C contains 5% Trichoraticectic acid, Group D contains 5% nitric acid, and Group E contains 5% formalin-nitric acid. Regardless of the specifics of the chosen decalcification solution, all procedures benefit from the inclusion of external stimuli. None of the variables were used in the current investigation; it was conducted only to compare various decalcifying chemicals. When time is not a concern, neutral EDTA may be recommended for preservation and presentation because of its ability to maintain soft-tissue integrity and provide high-quality staining. The formalin-nitric acid solution was one agent that appeared to strike a good compromise between speed and tissue preservation.

Chemical changes in root canal dentin of primary teeth after endodontic irrigation

Background: The current root canal irrigants used in dentistry are either antibacterial or decalcifying agents or their combinations. These agents can alter the chemical structure of dentin as well as change its permeability and solubility characteristics. Aim: The aim of this study was to evaluate the mineral content of root canal dentin after irrigating with five different endodontic irrigation solutions Materials and Methods: Sixty primary anterior teeth were selected for the study. Cleaning and shaping was done by step-back technique and divided randomly into six groups (n = 10): BioPure™ MTAD (Group 1), QMix™ 2-in-1 solution (Group 2), 0.9% normal saline (Group 3), coconut water (Group 4), and 5.25% NaOCl (Group 5) and the uninstrumented ten teeth were grouped as Group 6. After irrigation with final irrigants, the teeth were longitudinally divided into two halves. The levels of six elements calcium, phosphorus, oxygen, carbon, arsenic, and fluoride at the coronal and middle third in each specimen was assessed using scanning electron microscopy and energy-dispersive X-ray analysis. Statistical analysis was done by SPSS version 20 using one-way analysis of variance, and the comparison of means was conducted using post hoc Tukey test. Results: There was a statistically significant reduction in the calcium level at the coronal third of radicular dentin after irrigating with five different irrigants when compared with the uninstrumented teeth group. The phosphate level at the coronal third of the radicular dentin significantly increased in all the groups except for the saline group. The other elements such as oxygen, fluoride, and arsenic also showed significant differences in the coronal and middle levels after the irrigation. Conclusion: The endodontic irrigation by various irrigants changed the mineral levels in primary tooth dentin

A comparison of three decalcification agents for assessments of cranial fracture histomorphology.

The extraction of mineral calcium from bone by decalcification is a critical step in the preparation of histological samples for light microscopy. This study assessed the time required for complete decalcification and the resultant histomorphological preservation of bone histomorphology by three decalcification agents: 7% hydrochloric acid (HCl), 5% nitric acid, and 10% ethylenediaminetetraacetic acid (EDTA). The goal of this study was to identify which decalcification agent provides the optimal combination of expedient processing and quality histological outcomes of cranial fracture samples. HCl provided the most rapid decalcification ( =3.57 days), nitric acid followed closely ( =10.35 days), while EDTA took significantly longer on average ( =78.97 days) but encompassed a broader range of times. Decalcification agent, sample thickness, sample width, and decedent age are significant predictors of decalcification time. Sample visualization quality, measured for tissues, cells, and nuclei on a five-point Likert scale, was highest for samples decalcified in 10% EDTA, second highest using 5% nitric acid, and lowest for 7% HCl. The quality difference between EDTA and nitric acid was not highly significant for any of the three features. For basic assessments of bone histomorphology, the study results indicate 5% nitric acid is suitable for the decalcification of adult specimens and samples thicker than 3 mm. EDTA is a suitable agent for thin samples of the cranial vault (<3mm) from infants and young children less than three years old, decalcifying samples in a timeframe comparable to nitric acid while providing the best quality and clarity of samples.

Tooth decalcification using different decalcifying agents - A comparative study.

Background:In microscopic assessment of mineralized tissue, decalcification is an important step during tissue processing. The present study was attempted to compare the efficacy of various decalcifying agents and to evaluate the most efficacious decalcifying agent.Aims and Objectives:The aim was to study and compare the time taken for complete decalcification of the specimen by six different chemical agents; to study and compare the effect of various decalcifying agents on cellular and nuclear changes of hard and soft tissues; to study and compare the effect of various decalcifying agents used on the staining intensity with Ehrlich's Hematoxylin and Eosin stain and to determine the ideal decalcification technique.Materials and Methods:The six decalcifying agents, namely 5% nitric acid, 8% formic acid, formalin-nitric acid, 5% trichloroacetic acid, neutral ethylenediaminetetraacetic acid (EDTA) and Perenyi's fluid were used to decalcify 30 human permanent teeth (5 teeth in each solution). The endpoint of decalcification was evaluated by chemical (calcium oxalate test) as well as radiographic methods. The specimens were then subjected to processing, sectioning and staining with hematoxylin and eosin. The stained sections were observed under a light microscope and grading was done.Results:The results in the present study confirmed the fact that the time required for complete decalcification process was least in Perenyi's fluid, 5% trichloroacetic acid and highest in 14% EDTA. Teeth decalcified in 5% trichloroacetic acid, 8% formic acid, formalin-nitric acid and 5% nitric acid were easy to section. Sectioning was most difficult for teeth decalcified in Perenyi's fluid and 14% EDTA. The overall structure details as well as staining characteristics were best in teeth decalcified by 5% trichloroacetic acid and neutral EDTA and worst in teeth decalcified by Perenyi's fluid.Conclusion:Five percent trichloroacetic acid was showing the most efficient result as it balances both tissue integrity and time factor suggesting that it can be used as a stable decalcifying agent for routine histopathological diagnosis.

Acid-Based Decalcification Methods Compromise Genomic Profiling from DNA and RNA

BACKGROUND: Comprehensive genomic profiling (CGP) performed by next-generation sequencing of DNA detects genomic alterations including point mutations, insertions/deletions, copy number variations, and select gene rearrangements. When RNA sequencing is included in CGP, it allows for expanded detection of gene fusions, which are common in hematologic malignancies and sarcomas. When such tumors involve bone, a decalcification step is frequently employed to soften tissues prior to processing and sectioning. While commonly used acid-based decalcification methods work quickly, the resulting nucleic acid damage can be profound. In this study, we examine the effects of decalcification on DNA and RNA sequencing in the clinical setting. DESIGN: 1711 consecutive formalin-fixed paraffin embedded samples were evaluated by CGP during routine clinical care via DNA and RNA sequencing, using a hybrid-capture next-generation sequencing assay (FoundationOne®Heme). Specimen site [e.g. bone/ bone marrow or soft tissue] and decalcification status were extracted from pathology reports and H&amp;E review. Samples were considered decalcified if reported as such in the pathology report or if visible decalcified bone was present on the H&amp;E. Samples documented to be processed with fixatives other than formalin were excluded. Sequencing failures were defined as samples that failed DNA extraction (DNAx), RNA extraction (RNAx), or library construction (LC) due to insufficient nucleic acid to advance into sequencing. Samples were only evaluated for RNA if DNAx was successful (1594 cases). RESULTS: Specimen site was a strong predictor of sequencing failure, with a significant increase in failure rate from bone/bone marrow samples (n=619) compared to samples from soft tissue sites (n=1092) for both DNA (13.4% vs 4.6%, p=4.7E-9) and RNA (42.5% vs 13.5%, p&lt;2.2E-16). Of the bone/bone marrow samples, 237 of 619 samples were decalcified. Decalcification was associated with significantly higher failure rates than non-decalcified samples for both DNA (29.1% vs 3.7%) and RNA (67.4% vs 30.8%) (Table 2). One method of avoiding decalcification for bone marrow samples is utilization of clot preparations, where aspirates are processed as an FFPE block. Clot preparations fail sequencing significantly less often than decalcified core biopsies (DNA: 3.3% vs 18.8%, p=9.2E-06; RNA: 39.2% vs 70.4%, p=2.5E-03) (Table 3). CONCLUSIONS: CGP of samples acquired from bone and bone marrow sites is challenging, with a lower success rate for DNA and RNA sequencing than soft tissue sites. The higher overall failure rate correlates with use of decalcification agents leading to degradation of nucleic acids and impacts RNA sequencing significantly more than DNA (67.4% vs 30.8% failed). Clot preparations of bone marrow samples performed better than core biopsies for both DNA and RNA. The higher overall RNA sequencing failure rates still observed in in non-decalcified bone/bone marrow are predominantly due to RNA failure of non-decalcified clot preparations. These samples likely have increased failure rates secondary the use of non-standard fixatives (e.g. B+, Bouin's, AZF, etc.) not documented in the pathology report and the frequency of hypocellular clot preparations in conjunction with higher requirements for RNA yield compared to DNA yield. To increase CGP success rates, decalcification should be avoided when possible. Peripheral blood and bone marrow aspirate samples rarely fail sequencing (&lt;1%, data not shown) and are preferable to decalcified samples if adequate tumor is present. Bone marrow clot preparations perform better than bone marrow core biopsies and clot preparations should be fixed with 10% neutral buffered formalin. If decalcification is required for processing, EDTA based decalcification methods and/or minimizing decalcification times is recommended. Disclosures Duncan: Foundation Medicine, Inc.: Employment. Danziger:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Duncan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Hemmerich:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Edgerly:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc: Employment. Huang:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Vergilio:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Elvin:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. He:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Britt:Foundation Medicine, Inc: Employment. Reddy:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc: Employment. Sathyan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Alexander:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ross:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Brown:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ramkissoon:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Severson:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment.

The comparative analyses of decalcification procedures and methyl benzoate pre-treatment on tissue preservation and antigenicity in human acetabular labra.

The histological processing of musculoskeletal tissue might be challenging. The alteration of tissue composition e.g. by calcification of soft tissue in the elderly, after trauma or surgical interventions makes the histological processing of fixed tissue difficult. Additional steps of decalcification are then needed that probably affect the staining quality. In the present work, the effects of different decalcification agents and the intermedium methyl benzoate on histological staining methods and immunohistochemistry have been compared. Acetabular labra were fixed with 4% paraformaldehyde, left untreated or decalcified using 30% ethylenediaminetetraacetic acid (EDTA; Chelaplex®) or 6% trichloroacetic acid (TCA) for 1-4 days to investigate the effects of decalcification duration. Moreover, samples were pretreated with methyl benzoate or conventionally paraffin embedded independent of decalcification procedure and duration. The specimens were evaluated using hemalaun-eosin, Azur II- methylene blue staining or immunohistochemistry against ankyrin B to visualize nerve fibers. Decalcification with Chelaplex® or TCA reduced cutting artifacts without affecting the tissue morphology and proteoglycan staining but decreased antigenicity in immunohistochemistry. Interestingly, methyl benzoate further reduced cutting artifacts without altering tissue morphology and elevated antigenicity for Chelaplex® decalcified tissue samples in immunohistochemistry. The decalcification with Chelaplex® or 6% TCA preserves tissue morphology and proteoglycan staining similar to non- decalcified tissue but facilitates section processing. In immunohistochemistry both decalcification agents decreased antigenicity. Chelaplex® decalcified, methyl benzoate treated samples yielded an improved antigenicity.

Effect of decalcification agents on gene search using FFPE specimens

Effect of decalcification agents on gene search using FFPE specimens

Demineralization, Collagen Modification and Remineralization Degree of Human Dentin after EDTA and Citric Acid Treatments.

The aim of the study was to investigate the effects of several decalcifying agents used as irrigant solutions in endodontic treatment on collagen and mineral components of dentin. Coronal dentin discs from five caries-free human third molars with a smear layer were treated for one minute with a chelating solution (1% Ethylenediaminetetraacetic acid (EDTA), 10% EDTA, 17% EDTA, 10% citric acid). Mineralization degree (Ca/N and P/N atomic ratios, IR Iapatite/Iamide II and I1410(carbonate)/I554(phosphate) spectroscopic ratios) and possible collagen rearrangements (collagen infrared (IR) amide II e III shifts) were evaluated by environmental scanning electron microscopy (ESEM)/energy dispersive X-ray spectroscopy (EDX) and IR spectroscopy before and after treatment (T0) and after ageing (T24h and T2m) in simulated body fluid (SBF). At T0, analysis showed that the highest demineralizing effect was achieved using a 10% citric acid solution and 10% EDTA, while the smallest effect was observed when using 17% EDTA. No significant collagen modifications were detected upon treatment with 1% EDTA, while subtle changes were observed after the other treatments. At T24h or T2m, analyses showed the highest remineralization values for 1% EDTA and the lowest for 10% citric acid, mainly at T2m. The samples treated with 17% EDTA showed slight collagen rearrangements upon remineralization. In conclusion, the highest demineralizing effect was observed for 10% EDTA and 10% citric acid. Collagen rearrangement was found for all the treatments except for 1% EDTA. The highest remineralization capability in SBF values was recorded for 1% EDTA and the lowest for 10% citric acid. A slight collagen rearrangement upon remineralization was still present in 17% EDTA-treated samples. Clinical use as a chelating agent in the endodontic therapy of citric acid and concentrated EDTA solutions should be reconsidered.

Identification of degraded bone and tooth splinters from arid environments using palaeoproteomics

The analysis of the skeletal remains of vertebrates in archaeological contexts provides information about human-animal relationship and their environment. Their taxonomic identification based on macroscopic observation is not always possible due to fragmentation and poor preservation. In recent years, proteomics has emerged as an alternative but there is clearly a lack of data in arid environment where diagenesis rapidly affects the integrity of bone proteins. Here, we report the efficiency of three protocols for protein extraction. The protocols used harsh (1 M HCl and 0.6 M HCl) and soft (Tris-EDTA) decalcification agents and were tested on unidentified splinters from the 2000 years-old site of Toteng, Botswana. The preservation of the organic phase was first estimated using attenuated total reflectance Fourier transform infrared spectroscopy and a set of samples with contrasted collagen contents were selected for palaeoproteomics. The extracted proteins were submitted to a bottom-up proteomic approach involving trypsin digestion followed by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Our results identify Tris-EDTA buffer as the most suitable decalcification protocol for poorly preserved bones and propose a collagen content threshold of ~3% weight content for successful detection of peptides. This approach, combined with biogeographical and chronological repartitions of mammals in Africa allows refining taxonomic attributions for four out of nine splinters, leading to species identification. Data are available via ProteomeXchange with identifier PXD010725.

Comparison of two decalcification agents using microwave technology: a histochemical assessment of the rat cochlea

ABSTRACTDespite the prevalence of hearing loss, there are no FDA-approved chemical entities available for either otoprotection or treatment of hearing impairment. This lack of drug-based treatments has made the assessment of the middle and inner ear a rare, challenging exercise in the pharmaceutical industry. Recently, advances in the use of various decalcifying agents, as well as heating techniques utilizing microwave technology have proven to be effective at shortening processing times, minimizing tissue damage and potentially lowering the cost of histopathological evaluation and diagnosis. This study tested decalcification agents in order to determine the most efficient decalcification procedure for rat outer, middle, and inner ear structures while preserving tissue morphology and eliminating introduction of artifacts to best facilitate histopathological analysis. Presented here are the results of rat ear histological preparations using two decalcifying agents at increasing microwave times with heat controlled at the physiological temperature of 37 °C. In this analysis, an acid-based combination decalcification-fixative solution was found to significantly speed up the decalcification process when compared with the calcium chelator EDTA.

Analysis of the effects of several decalcifying agents alone and in combination with sodium hypochlorite on the chemical composition of dentine.

To investigate the effects of several decalcifying agents alone and in combination with sodium hypochlorite (NaOCl) on the organic and inorganic components of dentine using attenuated total reflectance in Fourier transform infrared spectroscopy (ATR-FTIR). Dentine slices from bovine teeth were submitted to (n=5) the following: 0.9% saline, 9% and 18% etidronic acid (HEDP), 5% and 10% tetrasodium EDTA (EDTANa4 ), 17% trisodium EDTA (EDTAHNa3 ), and 0.5% and 2.0% peracetic acid (PAA) for 0.5-10min; and to the combinations: G1 - mixture 5% NaOCl+18% HEDP (5 and 10min); G2 - mixture 5% NaOCl+10% EDTANa4 (5 and 10min); G2 - 2.5% NaOCl (5min)+17% EDTAHNa3 (1min); G3 - 2.5% NaOCl (5min)+0.5% PAA (1min); G4 - 2.5% NaOCl (5min)+9% HEDP (5min). Specimens of G2, G3 and G4 received final flushes with 2.5% NaOCl for 0.5-10min. Amide III/phosphate and carbonate/phosphate ratios of the spectra collected from the dentine specimens before and after immersion in the solutions were determined. Data were submitted to one-way repeated measures and one-way anova. For the same decalcifying agent, the higher the concentration and immersion time the greater the removal of phosphate, exposure of collagen matrix and consequently the increases in amide III/phosphate ratio. However, significant differences were found only between the two concentrations of PAA (P<0.05). PAA caused greater increases in this ratio, followed by EDTAHNa3 , EDTANa4 and HEDP, and this order was retained in the combinations with NaOCl. This ratio was significantly reduced in G1 (P<0.05) and not altered in G2 (P>0.05). Due to collagen degradation, the amide III/phosphate ratio reduced significantly after the use of NaOCl in G3, G4 and G5 (P<0.05). NaOCl required approximately 0.5s to deproteinate the collagen matrix exposed after phosphate removal by EDTAHNa3 and PAA. The carbonate of dentine was removed more rapidly than phosphate by all decalcifying agents alone and in G3, G4 and G5. In the combinations with NaOCl, the last irrigant used defined the dentine amide III/phosphate and carbonate/phosphate ratios. HEDP and EDTANa4 caused minor whilst EDTAHNa3 and PAA caused greater demineralization of dentine; both effects were time and concentration dependent. NaOCl degraded the dentine organic matrix more rapidly when it was exposed. Combinations of NaOCl and decalcifying agents can be used to create dentine surfaces with varying compositions for interaction with endodontic sealers.

A quantitative study of comparing the routine decalcification and microwave decalcification methods by using different decalcifying agents

Background: Decalcification is a process of complete removal of calcium salts from mineralized tissues. Microwave decalcification is a novel technique that can accelerate the duration of decalcification compared to the manual method, by increasing the temperature to evaluate the fastest decalcifying agents and methods (routine with Microwave decalcification) in both bone and tooth. Materials and Methods: The study group consisted of 80 hard tissues, 40 teeth and 40 pieces of 1 cm × 1 cm cut bone specimens were decalcified by both routine and microwave method using four decalcifying solutions (five bone pieces and five teeth in each agents for both routine and microwave method). The four decalcifying solutions were 8% nitric acid, 8% formic acid, 8%formal nitric acid, and 14% of ethylenediaminetetraacetic acid (EDTA). Results: Duration of decalcifi cation of bone and tooth by both methods with 8% nitric acid took 10 days for CD and 2 days for MD respectively. Similarly for rest of the acids took more time to decalcify in CD method than MD method. Fourteen percent EDTA gave excellent results followed by 8% formal nitric acid, and 8% formic acid has good results than 8% nitric acid. Conclusion: Microwave processing proved to be an efficient and reliable procedure for the decalcification of bones and tooth. Microwave decalcification yielded quicker and better results compared to the routine methods. Therefore, microwave can serve as a quicker and reliable diagnostic method for a pathologist.

Osteocalcin and Osteonectin Expression in Canine Osteosarcoma.

Osteosarcoma (OSA) is a malignant heterogeneous primary bone tumor responsible for up to 90% of all primary bone tumors in dogs. In this study, osteocalcin (OC) and osteonectin (ON) immunoreactivity was evaluated in 23 canine OSAs, 4 chondrosarcomas, 4 fibrosarcomas, 2 hemangiosarcomas, and 4 histiocytic sarcomas. The effects of three different decalcification agents (ethylenediaminetetraetic acid [EDTA], formic acid and hydrochloric acid [HCl]) on the immunoreactivity for OC and ON was also assessed. Immunoreactivity to OC was present in 19/23 (83%) cases of OSA and all cases of chondrosarcoma. In three OSAs the extracellular matrix showed immunoreactivity to OC. None of the fibrosarcomas, histiocytic sarcomas or hemangiosarcomas showed immunoreactivity to OC. The sensitivity and specificity for OC in canine OSA in this study was 83% and 71% respectively. For ON, 100% of both OSAs (23/23) and non-OSAs (14/14) showed cytoplasmic immunoreactivity to this antibody, giving a sensitivity of 100% but a complete lack of specificity. There were no significant differences in immunoreactivity for OC and ON between the different decalcification agents used. In conclusion, OC showed high sensitivity for identifying OSA but it failed to distinguish between OSA and chondrosarcoma, and the osteoid produced by neoplastic cells in most cases did not show immunoreactivity to OC. These factors may limit the practical utility of OC in the diagnosis of OSA in dogs when chondrosarcoma is a differential diagnosis. ON showed no specificity in detecting OSA and has little practical application for the diagnosis of OSA in dogs.

Comparison of three different techniques for histological tooth preparation

The histological processing of teeth is highly complicated because of containing both mineralized hard tissues and soft tissues. Depending on the type of decalcification agents used in processing, mild-to-severe deterioration in the tissue structure and inadequacies on clear staining of details by the histological stain may be observed. This study aims to compare the histological staining differences in the preparations from decalcified and undecalcified tooth roots by three different embedding materials and techniques. Following extraction, human single-rooted teeth crowns were cut off and roots were placed in 10% buffered neutral formalin. After fixation, roots were divided into two groups. One part of samples was decalcified in formic acid solution and the other was remained undecalcified. Decalcified roots were embedded in paraffin and glycol methacrylate (GMA)-based resin and undecalcified roots were embedded in methyl methacrylate (MMA)-based resin. Sections from all groups were stained with hematoxylin and eosin. The groups were compared in terms of general staining, brightness, density, density of the base stain, general morphology of cells, nuclear/cytoplasmic contrast, distinguish ability of pulp, odontoblast layer, predentin and dentin, preservation and traceability of dentinal tubule. In the preparations which were embedded into the MMA-based embedding material, an output lower than the paraffin group buthigher than the GMA-embedded group was provided. As a result, the best histological detail was obtained from the decalcified, paraffin-embedded sections.

Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid (DNA and RNA) recovered from bone biopsies

Molecular studies are part of standard care for cancer patients. Bone, a common and sometimes sole site of metastasis, requires decalcification for morphological examination. Many commonly used decalcification agents contain strong acids that degrade nucleic acids. The paradigm shift in oncology, with biomarker targeted therapy and gene expression profiling analysis, requires sufficient nucleic acid recovery from bone biopsy specimens. We systematically studied the effects of a spectrum of decalcification agents on the quantity and quality of RNA and DNA recovered from bone biopsies. Multiple bone biopsies of similar size and cellularity were fixed in 10% neutral-buffered formalin, randomized to various decalcification agents for 2 hours then processed, and embedded. Tissue lysates were obtained from unstained sections and nucleic acid isolated. DNA and RNA were quantified. Assessment of DNA and RNA integrity was accomplished by comparison of the average cycle threshold by polymerase chain reaction of selected housekeeping genes for each agent. Results were then analyzed by 2-sample t test. There was a significant decrease in both DNA and RNA yield and integrity with strong acids (hydrochloric, nitric) vs 14% EDTA and formic acid. DNA yield was (mean nanograms) 6.15 vs 68.68 (P<.001) and RNA was (mean nanograms) 121.53 vs 288.89 (P=.003), respectively. DNA integrity (mean cycle threshold) was 35.79 vs 30.16 (P<.001), and RNA was 33.03 vs 26.5 (P<.001), respectively. Decalcification of bone biopsies with EDTA or formic acid agents was associated with a significant improvement in recovered nucleic acid quantity and quality.

Evaluation and comparison of decalcification agents on the human teeth

Context:In routine histopathology, decalcification of bone and teeth is often an essential and important step during tissue processing. Various decalcifying agents have been used in the past. The rate of decalcification and the effect of decalcifying agents on the tissue and its staining characteristics are two important parameters which influence the selection of decalcifying solutions. Though some agents remove the calcium ions completely and rapidly, they adversely affect the staining characteristics and may also damage the organic components. There have been very few studies which have systematically evaluated the efficacy of these agents in decalcifying dental hard tissues.Aims:The present study was done to evaluate the rate of decalcification of six different decalcifying agents and also their effect on staining characteristics on dental hard tissues.Materials and Methods:Six decalcifying agents namely, neutral ethylene diamine tetra acetic acid (EDTA) decalcifying solution, 5% nitric acid, Perenyi's fluid, formalin–nitric acid, 5% trichloracetic acid, and 10% formic acid were used to decalcify 24 natural teeth (four in each solution). The endpoint of decalcification was evaluated by radiographic and chemical methods. The decalcified teeth were then routinely processed, sectioned, and stained with hematoxylin and eosin stains.Results:Neutral EDTA was the most considerate to the soft and hard tissues and 5% nitric acid was the least considerate to the tooth structure.Conclusions:Neutral EDTA, though being the slowest decalcifying agent among the six agents used in the study, gave excellent results for soft-tissue integrity, and best quality of both soft-tissue and hard-tissue stainings.

Macroscopic structure of the fin-rays and their annuli in pectoral and pelvic fins of Chinook Salmon, Oncorhynchus tshawytscha.

Light microscopy, enzyme clearing, and staining techniques were used to describe the structure of fin-rays in pectoral and dorsal fins of adult and juvenile chinook salmon,Oncorhynchus tshawytscha. In addition, several decalcification agents, fixatives, and staining methods were employed to demonstrate and determine the nature of the fin-ray annulus (yearly growth ring). Etched, transverse sections of fin-rays were examined by scanning electron microscopy (SEM). A description is provided of specific morphological regions along the length of fin-rays in both fins. Each ray consists of parallel halves, medial and lateral hemisegments, which diverge proximally and embrace underlying radial cartilages and are segmented distally. Fibrocartilage masses are associated with bone and hyaline cartilage in the proximal regions. There is evidence of periosteal, and perhaps endochondral, osteogenesis in the fin-rays. A novel fixation-staining method is described to depict the fin ray annulus in adult chinook salmon. Transverse sections of rays of adults, but not juveniles, demonstrate alternating narrow-dark and wide-light purple bands following prolonged decalcification/fixation in Bouin's fluid only with subsequent hematoxylin-eosin staining. The dark and light bands correspond to translucent (annuli) and opaque zones, respectively, in dried, unstained sections in transmitted light and in backscatter SEM. In conjunction with different light microscopic staining procedures, etched, transverse sections of fin-ray bone in SEM suggest that annuli are a manifestation of an ordered regionalization of the extracellular matrix of the bone in the fin-rays. J. Morphol. 239:297-320, 1999. © 1999 Wiley-Liss, Inc.

Effect of decalcification agents on immunoreactivity of cellular antigens.

The effects of several strong acids, weak acids, a proprietary decalcifier, and edetic acid on the immunohistochemical staining of cryostat and fixed paraffin embedded sections of tissue from a variety of normal and pathological calcified and uncalcified specimens were studied. Even decalcification in strong acids (HCl, HN03, 5% trichloracetic acid, HCl-edetic acid did not diminish the reactivity of many useful antigens (including leucocyte common antigen, intermediate filaments, S100 protein and epithelial membrane antigen). Weaker acids (formic acid, acetic acid) and edetic acid decalcified more slowly and generally showed greater preservation of antigenic reactivity with better morphology and staining quality. Trichloracetic acid was also useful as a quick one step fixation and decalcifying agent for both cryostat and routinely processed sections. Knowledge of the preservation of antigenic reactivity in decalcified tissue will be useful in the diagnosis of tumours of uncertain histogenesis and origin which affect calcified tissues.

PREVENTION OF EXPERIMENTAL RAT CARIES

In succession with the previous report, more detailed observation on two lesions, absent Pb-mark and infected tubulus in the fissure caries of rats, was reported in the present paper. Chronological observation of these lesions showed that the absent Pb-mark appeared earlier, being called the early lesion, and the infectious lesion was found at later stage of experiment in highest incidence, being called the late lesion, a successor of the early lesion.The early lesion often accompanied with half-moon shaped hematoxylin-phobe dentin matrix, brown pigmentation of dentin, bacterial crowd at the dentinoenamel junction, bacterial plaque in the fissure and bacterial invasion in the enamel binding above two bacterial settlements. The findings concluded that the once marked Pb-line in dentin did not remove spontaneously during 110 days after marking, but in vitro examination proved that removal of Pb-mark was easily resulted after action of decalcification agents, either acid or chelator. Therefore, the half moon shaped discoloration of the early lesion may probably be a track of decalcification. And decalcifying agents may sent from the bacterial crowd existing in the dentinoenamel junction. In dentin caries, however, the early lesion may follow the pre-early lesion characterized by the intact Pb-mark, the bacterial crowd and brown pigmentation.Instead of the early lesion, progress of infectious process in the late lesion may need action of neither acids nor chelators as bacterial products. The late lesion was often found under the cavity, exposing dentinal lesion toward the bottom of the fissure. In additional findings, at the summit caries, only the late type of lesion was found from the first. The differences between the early and late lesion in the mechanism of their production could be assumed from point of view on the different growth environment of bacteria existing, i-e. beneath the thick enamel layer and in dentinal tubulus under the open cavity where oxygen supply to bacteria may be much better than the dentinoenamel junction. Now carious progress in rat dentin may assume that in the early stage, after some lag phase of pre-early pictures, it proceeds exclusively decalcification by acid or chelator produced from bacterial crowd in the dentinoenamel junction, which in the later stage, invades into dentinal tubulus under the environment of better supply of oxygen and organic nutrient.