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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 sele...
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Background : Study of fibrilar, cellular and sub cellular structures of mineralized tissues is only possible after the removal of the calcium apatite of these tissues by the process of demineralization.
Aims: The present study aims to evaluate six commonly used demineralizing agents to identify the best decalcifying agent.
Materials and Methods: Th...
Background: Study of fibrilar, cellular and sub cellular structures of mineralized tissues is only possible after the removal of the calcium apatite of these tissues by the process of demineralization. Aims: The present study aims to evaluate six commonly used demineralizing agents to identify the best decalcifying agent. Materials and Methods: The...
Citations
... An ideal agent should also facilitate a reasonably rapid decalcification process while minimizing tissue damage or distortion. It should also be easy to handle, ensuring safety for laboratory personnel and compatibility with subsequent staining techniques (Sanjai et al. 2012). ...
... Each agent has its advantages and limitations, demanding a balance between speed, preservation of tissue structures, and the desired analyses in bone decalcification for research or clinical purposes. The speed of decalcification and the impact of decalcifying agents on tissue and staining properties are crucial factors that affect the choice of decalcification solutions (Sanjai et al. 2012). Faster agents such as nitric acid can damage tissue, affecting staining techniques. ...
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.
... Ali et al (4) advocated for the application of 10% HCOOH as the superior DA when compared to 5% trichloroacetic acid in accordance with the results of the present study. Sanjai et al (14) conducted a comparative analysis of six DAs, finding 5% HNO 3 to be the swiftest, neutral EDTA to be the slowest, and neutral EDTA to yield superlative outcomes in terms of soft-tissue integrity, while 5% HNO 3 yielded the least favourable outcomes. Sangeetha et al (15) juxtaposed customary decalcification against microwave-assisted decalcification using 5% HNO 3 , 5% HCOOH and 14% EDTA with regard to expedition, the preservation of tissue architecture and the efficacy of staining. ...
... With this, the molars, which are the largest and heaviest teeth, needed five days (≅120 hours) to completely dissolve, whereas the incisors, which are the lightest teeth, took three days (≅72 hours). [13] Even though there was a substantial difference in the weight of the canines and premolars between the maxillary and mandibular arches (P < 0.01), the time required to reach the endpoint of decalcification remained the same, at around 4 days. ...
Introduction: Immunohistochemistry (IHC) has not always been an easy field for the research beginners like
postgraduates, research fellows and scientists. Meaningful interpretation of IHC positivity needs expertise.
This could be made easier for beginners by developing a conceptual framework of markers. The literature
review revealed a lack of qualitative evidence on the hitherto IHC studies on oral diseases about the overall
expression of IHC markers and its comparison with pathology and normal tissues.
Aim: This scoping review aimed to examine the literature and classify the various immunohistochemistry
markers of oral diseases based on the tissue, cell and site of expression.
Materials and Methods: The review was in accordance with Preferred Reporting Items for scoping
reviews (PRISMA -ScR). Electronic databases such as PubMed and Cochrane were searched for relevant
articles till 2021.
Results: We included 43 articles. We found five different possibilities of the site of expression of a
marker in a cell. They are the nucleus, cytoplasm, cell membrane, extracellular matrix or any of the above
combinations. Based on the tissue of expression, we also mapped the markers expressed in oral diseases to
their tissue of origin as ectoderm, endoderm, mesoderm and markers with multiple tissues of expression.
Based on our results, we derived two classifications that give an overview of the expression of IHC markers
in oral diseases.
Conclusion: This scoping review derived new insight into the classification of IHC markers based on cell
lineage, tissue and site of expression. This would enable a beginner to better understand a marker with its
application and the interpretation of the staining in research. This could also serve as a beginner’s guide
for any researcher to thrive and explore the IHC world.
Keywords: Biomarkers, classification, immunohistochemistry, markers, oral disease
... El punto final de la descalcificación se realizó con base en los resultados de tres técnicas de verificación: controles radiográficos del creciente aumento de la hipodensidad, empleado por autores como Castania 33 y Choube, 34 en estudios histológicos, control de dureza, utilizado por autores como Sanjai et al, 35 Gupta et al, 36 que denotaba la flexibilidad que adquirían las raíces de los dientes como consecuencia de la pérdida de iones calcio y fósforo por exposición a los descalcificantes; y el control de Robertson que evidenciaba la pérdida de esmalte dental de los dientes en cada control. 37 Para la fase de deshidratación de los tejidos, la literatura muestra que la sustancia más utilizada es el alcohol etílico, y se usa en concentraciones graduales para evitar distorsión del tejido debido a su efecto de contracción, proceso que se realizó en este trabajo. ...
RESUMEN Antecedentes: La Diafanización dental es una técnica que permite transparentar los dientes haciendo visible la anatomía interna de los conductos radiculares ofreciendo una herramienta pedagógica económica y confiable; sin embargo, la literatura no reporta protocolos estandarizados para obtener una diafanización dental predecible. Objetivo: Obtener un protocolo estandarizado para diafanización dental, como modelo educativo, a partir de la revisión de la literatura y la realización de un estudio piloto. Materiales y Métodos: Se realizó una búsqueda sistemática en las bases de datos Scopus y Medline con los términos Mesh "root canal", "diaphonization", "clearing", "morphology" anatomy", y se estructuró una tabla de extracción con las variables más representativas para establecer las 3 fases de la Diafanización, a. Descalcificación, se evaluó Ácido Nítrico 5% (HNO3), Ácido Fórmico 10% (CH2O2) y EDTA 10%, b. Deshidratación, se empleó Alcoholes etílicos ascendentes, c. Clarificación, se evaluó Metil Salicilato y Aceite de Inmersión Sintético. Se seleccionaron 54 dientes, 36 sin endodoncia, y 18 con endodoncia, y se distribuyeron en dos grupos: Grupo A. Dientes sin endodoncia, Grupo B. Dientes con endodoncia, constituidos por 18 subgrupos que estaban definidos de acuerdo al tipo de descalcificante, momento de aplicación medio de contraste y medio de Clarificación. Resultados: El Ácido Nítrico al 5% pese a que fue el más corrosivo, permitió el mayor flujo y accesibilidad para el medio de contraste (Tinta China) en especímenes sin endodoncia. De igual manera, el Ácido Fórmico al 10%, preservó la estructura de los dientes tratados endodónticamente. El Metil Salicilato como clarificante, brindó mejores resultados visuales alcanzando una mayor transparencia. Conclusiones: El desarrollo de un estudio piloto para estandarizar técnicas de diafanización en odontología, permite la estructuración de un protocolo educativo que posibilita conocer la gran variabilidad anatómica de los dientes y la comprensión y análisis de los dientes que han sido tratados endodóncicamente, aportando una herramienta pedagógica para la comprensión de la anatomía radicular. El uso de Ácido Fórmico al 10%, en dientes con tratamiento de endodoncia y de Ácido Nítrico 5% en dientes sin endodoncia, con una transparencia alcanzada por el uso del Metil Salicilato, muestran los mejores resultados visuales en anatomía y obturación endodóntica. PALABRAS CLAVE Aclaración ABSTRACT Background: Dental diaphonization is a technique that allows the teeth to be made transparent, making the internal anatomy of the root canals visible, offering an economical and reliable pedagogical tool; however, the literature does not report standardized protocols to obtain a predictable dental clearance. Aim: obtain a standardized protocol for dental diaphonization as an educative model from the review of the literature and the realization of a pilot study. Materials and methods: A systematic search was made on databases Scopus and Medline, with the Mesh terms "root canal", "diaphonization", "clearing", "morphology" and, "anatomy", and an extraction table was structured with the most representative variables to establish the three diaphanization phases, a. Decalcification, 5% Nitric Acid, 10% Formic Acid (TBD-2) and 10% EDTA were evaluated, b. Dehydration, ascending Ethyl Alcohols were used, c. Clarification, Methyl Salicylate, and Immersion synthetic oil were evaluated. 54 teeth were selected, 36 without root canal treatment and 18 with root canal treatment, then they were distributed into two groups: Group A, Teeth without root canal treatment, and Group B, Teeth with root canal treatment. Each group was constituted of 18 subgroups defined in order of the decalcification agent type, moment of the contrast medium application, and clarification agent type. Results: Even though 5% Nitric Acid was the most corrosive agent, it allowed a better flow and accessibility for the contrast medium (Chinese ink) in teeth without root canal treatment. Likewise, 10% Formic Acid preserved the structure of the endodontic tooth. As a clarification agent, the Methyl Salicylate showed better visual results, achieving greater transparency. Conclusion: The development of a pilot study aimed to standardize diaphonization techniques in dentistry allows the structuring of educative protocols that permit knowing the great tooth anatomic variability and the comprehension as well as the analysis of the root canal treated teeth, contributing to a pedagogic tool for the root anatomy awareness. Using 10% Formic Acid on root canal-treated teeth and 5% Nitric Acid on root canal not treated teeth, with transparency achieved by using Methyl Salicylate, showed better visual results on anatomy and endodontic filling.
... However, the effect of the decalcifying agents also depends on various factors including concentration used, temperature, time duration for decalcification, tissue suspension, size and type of mineralized tissue. 1,7 The purpose of this study was to evaluate qualitative histological features of human premolar teeth using various decalcifying agents. Dental hard tissue and soft tissue including dentin, cementum, and dental pulp tissue were histologically evaluated. ...
... The teeth were periodically checked, and the end point of decalcification was estimated by physical and radiographic methods. 1,9 The physical method was performed by bending and probing the tooth using a fine needle. When the needle passed through the whole tooth thickness, the decalcification process was then over. ...
... This prevents tissue collapse and preserves tissue architecture superior to acid decalcifying agents. 1 Regardless of decalcification time, our findings pointed out that EDTA was suitable for decalcification for histological examination of both dentin and cementum. Although no statistical difference was observed for the histological score of dentin among the chemicals except the formic acid, EDTA tended to preserve dentin morphology better than the others. ...
Objective Dental hard tissue is among the hardest tissue of humans because it contains high amounts of inorganic substances. This leads to difficulty in preparing histological sections for histopathological examination. Acid and chelating agents are generally used to decalcify teeth. We aimed to compare the histological quality of teeth decalcified with various calcifying agents including 5% nitric acid, 50% formic acid with 20% sodium citrate (Anna Morse solution), 10% formic acid, commercial solution, and 14.4% neutral EDTA.
Materials and Methods Freshly extracted premolar teeth were fixed and submitted for decalcification using different agents. Histological examination was qualitatively evaluated for tissue integrity and staining quality.
Results Dentin integrity of teeth decalcified with all decalcifying agents did not show any statistical differences except that with the formic acid, whereas cementum integrity decalcified with neutral EDTA showed a superior score compared with other agents. Tissue integrity and staining quality of dental pulp cells were the best decalcified with neutral EDTA or Anna Morse solution.
Conclusion Our findings demonstrated that EDTA and Anna Morse solution gave a similar efficiency in the preservation of tissue integrity while Anna Morse solution may be recommended as a decalcification agent in routine use due to the more satisfying decalcification time than EDTA.
... 6 EDTA solution and Von Ebner's solution was replaced after every 5 days. 7 The status of decalcification was evaluated with weight change, followed by chemical and radiographic confirmation method. Periodic weight loss was weighed and weight loss percentage was calculated and tooth were sectioned, stained and examined under microscope for evaluation at each set interval of weight loss. ...
The histopathological examination of mineralized tissues requires decalcification of teeth which is an essential and important step during tissue processing. In the present study we attempted to decalcify teeth using strong and weak acids and a chelating agent with various methods to identify completion of decalcification along with the observance of weight loss percentage of a tooth.
Aim
To compare decalcification with conventional decalcification method with strong acid, weak acids and a chelating agent with respect to preservation of tissue structure, efficacy of staining in association with weight change.
Materials and methods
A total of 64 multi-rooted and single rooted teeth were used, with group of 16 teeth, (16 molars, 16 pre-molars, 16 canines and 16 incisors) for each of the solution as Chelating agent (10% EDTA), Strong acid(10%HNO3), Weak acid (5% Tri Carboxylic acid) and Von Ebner's solution (hydrochloric acid & sodium chloride), were used in the study respectively. The efficacy of decalcifying agents was evaluated by recording the time taken by particular acid to decalcify the tooth completely and the weight change was observed at set intervals till the completion of decalcification. The endpoint of decalcification was also confirmed with radiographic and chemical methods. The decalcified teeth were then routinely processed, sectioned, and stained with haematoxylin and eosin stains. Different methods were used to confirm the completion of decalcification. After decalcification, all the teeth were examined macroscopically and microscopically.
Results
At 70–80% of weight change of a tooth decalcification is complete. 10% EDTA was best suited to the soft and hard tissues in comparison to other solutions. 5% TCA was fair in staining quality and maintenance of hard tissue structures was satisfactory to 10%HNO3 and Von Ebner's solution.
Conclusion
The final impression led to the proposition that EDTA was indeed the best decalcifying agent available if the results required are not urgent. For situations where time constraint is there, 5% Tri Carboxylic Acid can be used.
... [15,16] Hence, the current study is innovated by refining its outcome to understand the chemical means of dissolution of pulp stones to achieve a more qualitative outcome. A pilot study was done with various decalcifying agents like 5%, 10% nitric acid, and 5% formic acid, 1% acetic acid, [13,17,18] potassium citrate, magnesium citrate, [19] and Udiliv gel. [20] However, these agents demonstrated inadequate as well as a longer period for decalcification of pulp stones. ...
... The three reagents of PSDA were chosen based on the following criteria that it must decalcify at a reasonable speed, ensure complete removal of calcium, and cause minimal damage to surrounding tissues. [13,17,18] The solution majorly contained hydrochloric acid -an agent commonly used for decalcification that produced better results when compared to formic acid and nitric acid. It has also got excellent soft-and hard-tissue integrity. ...
... It has also got excellent soft-and hard-tissue integrity. [13,17,18,21] Potassium hydrogen phthalate -an acidic salt compound that acts as a buffer and stabilizes the pH of the reagents, [22] and dimethyl sulfoxide -an inert solvent which acts as a penetration enhancer. It is also shown to improve the immediate and long-term bond strength of dentin. ...
Background:
Despite constant advances in science, obscurity remains in the efficient removal of pulp stones to aid in successful root canal treatment. In this context, chemical means of dissolving pulp stones were explored.
Aim:
The aim of this study is to evaluate and to compare the efficacy of decalcifying agents on the dissolution of pulp stones.
Materials and methods:
The study was divided into two groups for pulp stone analysis (21 samples) and dentin analysis (54 samples). Twenty-one pulp stones from patients aged 18-70 who underwent root canal treatment were collected and divided into three subgroups (n = 7) randomly. They were subjected to chemical treatment in a labeled glass container with 5 ml of the respective chemical agents, such as 17% ethylenediaminetetraacetic acid solution (positive control), no treatment (negative control), and newly developed Physiological Simulated Decalcifying Agent (PSDA). At the end of the study period (24 h), the samples were removed, rinsed with deionized water, and subjected to physical analysis, scanning electron microscopy (SEM), and Energy -dispersive X-ray spectroscopy (EDS) analysis. Under dentin analysis, 54 maxillary premolars scheduled for orthodontic extraction without caries or extensive restorations were selected, following which 2-mm thick transverse dentinal sections at the cementoenamel junction level were obtained and randomly divided into two groups for SEM (n = 21) and microhardness analysis (n = 33). The samples were subjected to respective chemical treatment groups similar to pulp stones for 24 h and analyzed using SEM, EDS, and microhardness analysis.
Results:
Postchemical treatment with the newly developed decalcifying solution, the pulp stones showed the absence of nodular crystallites and surface softening under SEM and a decrease in the calcium level under EDS analysis. Concerning the microhardness of dentin, no significant changes could be observed.
Conclusion:
The newly explored PSDA was found to be efficacious in the decalcification of pulp stones at a clinically relevant time of 24 h, without significantly affecting the structural integrity and the hardness values of dentin.
... Beyond the chemical mode of action, solution factors such as pH and temperature, sample thickness, and method of demineralization (i.e., immersion, microwave, sonication, etc.) also influence the length of decalcification [1,[4][5][6]. Overall, strong acids are the most expedient, requiring only a few days [4,[7][8][9][10][11][12], while bone submerged in a chelating agent such as EDTA may take several months to fully decalcify [1,4,8,9,[13][14][15]. Under normal circumstances, forensic case reports are required to be completed in 30 to 60 days; thus, processing time is a decisive factor in the substance chosen for demineralization. ...
... Numerous studies have performed comparative analyses of histological preservation by decalcifying agents, with a general consensus that slow decalcifications in EDTA provides superior outcomes for staining and cell and tissue morphology at the cost of lengthy immersion times [1,4,7,[11][12][13]15,16]. However, these inquiries are predominately based on animal models [4,5,7,8,11,[16][17][18] and human dentition [9,13,15], rather than human cranial bone. ...
... Numerous studies have performed comparative analyses of histological preservation by decalcifying agents, with a general consensus that slow decalcifications in EDTA provides superior outcomes for staining and cell and tissue morphology at the cost of lengthy immersion times [1,4,7,[11][12][13]15,16]. However, these inquiries are predominately based on animal models [4,5,7,8,11,[16][17][18] and human dentition [9,13,15], rather than human cranial bone. Furthermore, exhaustive literature searches yielded no available standards for the decalcification of healing bone. ...
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 (X¯ = 3.57 days), nitric acid followed closely (X¯ = 10.35 days), while EDTA took significantly longer on average (X¯ = 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 (<3 mm) 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.
... They also concluded that minimal shrinkage of soft tissue and minute tissue loss is shown by 10% formic acid. [19] Singh S and Sicar K (2010) conducted a study and they have demonstrated that preservation of morphological characteristics is based on staining uniformity. The various decalcifying agents used in this study were as follows-10% of formal formic acid, 10% of formal nitric acid, and 5% of formal EDTA. ...
Background: In bony specimens, elimination of calcium is obtained by a method known as “Decalcification”. It is completed through the means of chemical agents such as acids, chelators etc that combine with ions of calcium. Decalcifying agent is used in regular conventional method where the hard tissue is placed at a room temperature (20-25°C) with modifications of the solution at orderly intervals until the final cutoff point is obtained. Usage of microwave oven for the process of decalcification is a new and fast method in contrast to the routine conventional method of decalcification. In this study, an attempt has been made to regulate and compare the conventional procedure of decalcification with decalcification done by microwave oven of hard tissue specimens by using nitric acid of 10% concentration with regards to decalcification speed, conservation of tissue architecture as well as productiveness of staining. Objectives: The study will made a comparison of Conventional and Microwave Bone Decalcification Methods by using 10% Nitric Acid. Methodology: This prospective analytical study, will include decalcification of 30 hard tissue specimens by microwave method and conventional method. The results will be compared in terms of decalcification speed, conservation of tissue architecture and staining productiveness. Necessary tests will be applied to analyze the data. Expected Results: Significant advantages of microwave method are expected over conventional method of decalcification. Conclusion: The conclusion will be drawn based on careful analysis of the results.
... Some mineral acids, such as nitric acid, hydrochloric acid, and weak organic acids such as formic acid, were used in histological decalcification [141,[154][155][156]. They can achieve fast decalcification, but would induce damage to the bone tissues [157][158][159]. ...
... The chelating reagents, such as ethylene diamine tetraacetic acid (EDTA), can also be used for decalcification. EDTA causes little damage to tissue and can preserve the activity of antigens and certain enzymes in tissues, which make it suitable for subsequent immunochemistry [157][158][159]. It has been utilized in the clearing methods such as PACT-deCal [160], Bone-CLARITY [161], PEGASOS [82], etc., for bone clearing or whole-body clearing. ...
