Decalcification Solutions Research Articles

Effect of decalcification on immunohistochemical staining of trichorhinophalangeal syndrome type 1

AbstractTo evaluate the effect of different decalcification solutions on the immunohistochemical staining of trichorhinophalangeal syndrome type 1 (TRPS1), and to provide a reliable basis for the accurate diagnosis of bone metastasis of breast cancer. Due to the limited biopsy samples of bone metastatic cancer, 20 cases of invasive breast cancer were selected to simulate bone metastatic biopsy, dividing into four groups: undecalcified group, 30% formic acid group, 10% hydrochloric acid group and 10% nitric acid group. Immunohistochemical staining was performed after treatment for 2, 6, 18 and 24 h. There was no change in the proportion and intensity of TRPS1 cells in the formic acid decalcification group within 18 h, but decreased significantly after 24 h. The staining intensity of TRPS1 and the proportion of stained cells were significantly decreased in the hydrochloric acid decalcification group since 2 h treatment. The percentage and intensity of positive cells in the nitric acid decalcification group changed little or no change within 6 h, and then gradually decreased with the extension of time. Another three invasive breast cancer samples were used to compare the effects of different decalcification solutions on the same case. In brief, we conclude that the effect of 30% formic acid decalcification on TRPS1 staining is small when the time is less than 18 h. 10% nitric acid decalcification should be controlled within 6 h, which has little effect on TRPS1 staining, and 10% hydrochloric acid decalcification will lead to significantly less positive TRPS1 staining, so it is not recommended for breast bone metastasis tissue decalcification.

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

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.

Introduction of a decalcification method for bone marrow biopsy tissue

Bone marrow biopsy is one of the important means of hematopathological diagnosis, which has decisive diagnostic significance for various benign and malignant lymphohematopoietic system diseases. Its diagnostic value includes morphological observation, immunohistochemistry, genetics, and molecular biology testing. Owing to the unique nature of bone marrow biopsy, decalcification is an essential step in the pre-treatment process. Its purpose is to remove calcium from bone tissue, preserve intact collagen fiber components, facilitate tissue sectioning, and prevent tissue detachment during staining. If bone marrow biopsy lacks sufficient decalcification, preparing a section is difficult. Conversely, if decalcification is excessive, it can seriously disrupt tissue antigen activity. Therefore, a decalcification method with high decalcification efficiency and mild antigen damage is essential for bone marrow biopsy. This article introduces a bone marrow biopsy tissue decalcification method with high efficiency and less antigen loss: decalcification is performed at room temperature with 12% formic acid and 8% hydrochloric acid decalcification solution on a shaker.

Novel processing and staining methodology of bottlenose dolphin (Tursiops truncatus) teeth for age determination

ABSTRACT Age determination of bottlenose dolphins (Tursiops truncatus) is a critical tool in understanding both individual and population health. There are many methods of aging bottlenose dolphins including analysis of teeth, pectoral flipper radiographs, and epigenetics. The most common and oldest method for aging toothed cetaceans is the counting of growth layer groups (GLGs) in the teeth. Current techniques have technical and repeatability challenges. Therefore, a processing technique that results in better resolution of GLGs is needed. This study compares different decalcifications and different histochemical staining techniques. Decalcification was done using 10% EDTA, Kristensen’s decalcification, and Rapid Decalcification Solution (RDO). Following decalcification and routine processing, GLGs were assessed using Hematoxylin and Eosin (H&E), hematoxylin, Giemsa, Wright-Giemsa, Toluidine Blue (T-Blue), Masson’s Trichrome, and Congo Red staining techniques. Decalcification with Kristensen’s and staining with Masson’s Trichrome and Congo Red were determined to best highlight GLGs. This processing and staining was then applied to a sample population of 102 bottlenose dolphins that were evaluated independently and blindly by two observers. Of the 102 dolphin samples, 13 (12.7%) were unable to age due to no clear distinction or distortion between GLGs.

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.

Определение сроков декальцинации образцов грудины, коленного сустава и препарата носовых ходов мышей, песчанок и хомячков

Procedure of decalcification necessary for histology of bone tissue. Decalcification is the procedure of removing minerals from the bone organic matrix. Solutions of varying acids usually used as a decalcification solution. Whole bone or piece that had been cut out preliminary are can be sampled for decalcification. Study of a knee joint and nasal cavity specimens usually uses in preclinical research. Also, for the study of bone marrow, a sternum often is sampled. The cutting out of small piece often leads to the destruction of the sample, because of their small size. Thus, whole maxilla, whole sternum and knee joint to include femur, fibula and tibia are more often sampled for research. This article describe a time of decalcification above-mentioned specimen that were sampled from mice, gerbils and hamsters. Solutions of formic and hydrochloric acids were used as the decalcification solution. The decalcification solution were changed every other day. The severity of decalcifications and specimen readiness for histology procedure were evaluated by gravimetric method. We found that the trends in mass change and decalcification time change were the same for all animal types noted above. In none case, the absolute severity of decalcification was reached, within 10 days. However, the severity of decalcification of whole specimens of bone was sufficient to make a quality histological slide. All specimens achieved the sufficient severity of decalcification on 4th day. This period allows to evaluate the morphology of the respiratory and olfactory epithelium of the nasal cavity, joint cartilage and bone marrow cells. The period of decalcification can be extended to 7 days, if anatomic and topographic features and them the best quality more important. The period of decalcification more 7 days leaded to irreversible change of cell morphology. Cells had poor detalization, cell nuclei were stained with hematoxiline badly, cell borders were unclear.

Abstract P6-04-17: High Intra- and Inter-block concordance of HER2 immunohistochemistry (IHC) scores across breast cancer samples and impact of decalcification procedures

Abstract Background & Objective Trastuzumab deruxtecan is effective in HER2-positive (IHC3+ or ISH-positive) and HER2-low (IHC1+; IHC2+/ISH-negative) breast cancer. We assessed consistency of HER2-positive and HER2-low categorization by IHC across and between breast cancer samples with a range of HER2 expression. The impact of decalcification procedures on HER2 IHC staining was also assessed to determine whether HER2 expression is likely to be compromised in decalcified samples from metastatic bone disease. Methods Three non-consecutive tumor sections (approximately 100 μm apart) from 50 commercially obtained breast cancer resections (2 blocks/resection) and 20 biopsies (sections ~40 μm apart) representing a range of HER2 IHC expression, and with known ISH status, were stained using the VENTANA anti-HER2/neu 4B5 IHC assay, scored per ASCO-CAP (2018) guidelines and intra-block/inter-block concordance reported for HER2-positive and HER2-low categorization. To investigate the impact of decalcification, four resection samples were formalin-fixed, divided into four pieces, of which three were pre-treated with either 10% formic acid, Decal STAT or Richard Allan Scientific< sup>TM Decalcifying Solution before processing and staining with the 4B5 IHC assay. Results A single pathologist reviewed all samples. Intra-block agreement for HER2-low (proportion of blocks in which 3 of 3 sections had identical status) was 89.5% for biopsies (17/19) and 80.6% for resection samples (n=79/98). Inter-block agreement (proportion of resections in which two independent blocks had the same majority status for HER2-low) was 91.7%. Intra and inter-block agreement for HER2-positive was >90%. Exposure to Decal STAT or Richard Allan Scientific< sup>TM Decalcifying Solution caused a significant reduction in HER2 IHC1+ and IHC2+ staining in breast cancer samples. 10% formic acid had no apparent impact on HER2 IHC staining in all four samples tested. Conclusion There was high HER2-positive and HER2-low agreement within breast cancer biopsies and resection blocks and highly consistent HER2-positive and HER2-low categorization between different blocks from tumor resections. Our results suggest HER2 staining heterogeneity does not impact determination of HER2-positive and HER2-low status in the majority of cases. Accurate assessment of HER2 IHC1+ and IHC2+ status was observed to be compromised by Decal STAT and Richard Allan decalcification solutions. Decalcification of bone metastasis samples for HER2-positive and HER2-low status determination may be possible using formic acid, but this would require further validation. Citation Format: Asimina Tsirka, Charo Garrido, Marietta Scott, Paul Scorer, Anne-Marie Boothman, Tsuyoshi Matsuo, Craig Barker. High Intra- and Inter-block concordance of HER2 immunohistochemistry (IHC) scores across breast cancer samples and impact of decalcification procedures [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-17.

Idiopathic calcinosis cutis with unusual histomorphology and negative von Kossa stain: A diagnostic pitfall

Introduction: Calcinosis cutis is characterized by basophilic crystalline or amorphous calcium within the dermis/subcutis. Calcinosis cutis has many different clinical subtypes and associated etiologies that may lead clinicians to consider this diagnosis. Von Kossa and/or Alizarin red special stains may be used by pathologists to highlight deposition of insoluble calcium salts, especially in histologically ambiguous or subtle cases. Case Report: We report the case of a 27-year-old male who presented with an inferior right buttock mass clinically diagnosed as epidermal inclusion cyst(s). Gross pathology revealed a gray-white pasty substance within a cyst-like structure. Histopathologic examination demonstrated pools of amorphous blue-gray material with rare deposits of admixed coarse calcifications surrounded by foreign-body giant cells. No cyst wall was seen in the specimen. Special stains, including von Kossa, were initially negative. Following additional review, it was discovered that surface decalcifying solution had been applied to the paraffin block in the histology lab prior to microtome sectioning. Hypothesizing that this could be the cause of the unusual morphology, the paraffin block was reprocessed and subsequent H and E stained sections displayed characteristic basophilic calcium deposits, which were correspondingly positive by von Kossa stain. Conclusion: The histopathologic diagnosis of calcinosis cutis is apparent by HandE in most cases, though von Kossa and/or Alizarin red special stains can be used to aid the pathologist. Given the unusual histomorphology following surface decalcification and initial lack of von Kossa stain prior to reprocessing, this report serves to make pathologists aware of this potential diagnostic pitfall.

Comparative and qualitative analysis of various decalcifying agents and their affect on staining and texture of the permanent extracted human teeth

Teeth are among the hardest human tissues, because they are composed of large amounts of inorganic compounds. Teeth are very difficult to prepare for microscopic examination. Chemical Acids and chelating agents traditionally have been used to remove inorganic contents. We compared various decalcifying agents including strong acids, weak acids, chelating agents. Freshly extracted teeth were fixed and decalcified using formic nitric acid, formic acid, nitric acid, EDTA, trichloracetic acid and Perenyi’s fluid. Decalcified teeth were evaluated histologically for tissue preservation and staining characteristics. The study aimed at analyzing the rapidity and affects the tissue integrity and staining properties of decalcification of tooth with various decalcifying agents. 60 freshly extracted human teeth were divided into 6 different groups and each was treated with varying concentrations of decalcification solution until they were completely decalcified. Teeth decalcified with nitric acid solution resulted in rapid decalcification compared to other acids. Teeth treated with EDTA showed very minimal removal of inorganic content over a very long period of time. In routine practice 5% nitric acid can be effectively used for rapid decalcification of teeth in the histopathology laboratory however best staining qualities were noted among the teeth decalcified with formic acid and EDTA.

To estimate the effectiveness of different decalcifying solutions by electrolysis

To estimate the effectiveness of different decalcifying solutions by electrolysis

Evaluation of EDTA and nitric acid solutions for decalcification of joints in AG/WT, BALB/c, C57, DBA1/J mice, and in Wistar rats

ABSTRACT Decalcification of mineralized samples for microscopic analysis involves competing factors including decalcification time, preservation of tissue integrity and cost. We investigated the utility of different decalcification solutions for studying joints in AG/WT, BALB/c, C57, DBA1/J mice and Wistar rats. The hind paws of the rodents were removed and fixed with 10% buffered formalin. Specimens were divided randomly into three groups for demineralization: 10% nitric acid, 12.5% EDTA at room temperature and 12.5% EDTA at 35 °C with shaking. Sections of joints were stained with hematoxylin and eosin (H & E). We evaluated decalcification time and expense, ease of cutting sections, preservation of nuclear basophilia and intranuclear detail, and intensity of eosin staining. The 10% nitric acid solution produced the most rapid decalcification for the mice, but not the rats. The 12.5% EDTA solution at 35 °C with shaking did not decrease decalcification time. Effects on microtomy were variable as were the effects on H & E staining. The EDTA solution provided the best basophilia and intranuclear detail for the mice. For rats, only 12.5% EDTA at 35 °C with shaking produced good preservation. Preservation of nuclear basophilia and intranuclear detail for rats was best with 10% nitric acid and EDTA 35 °C. For mice, 10% nitric acid failed to preserve nuclear basophilia and intranuclear detail. For intensity of eosin staining, EDTA at room temperature and EDTA 35 °C was best for both mice and rats. Sections also exhibited good H & E staining in most samples decalcified with 10% nitric acid. Although we found considerable variation among groups of animals, we found less variation among the different mouse strains than between mice and Wistar rats.

Optimization of decalcification techniques for histologic examination of the rat maxillary and mandibular incisors for toxicity studies

ABSTRACT The objective of this study was to provide optimized processing for examination of rat incisors in nonclinical toxicity studies that enables analysis using immunohistochemistry (IHC). Rat maxillas and mandibles were decalcified in Immunocal, a formic acid decalcifier, and Decal Stat, a hydrochloric acid decalcifier, to evaluate tissue quality when with hematoxylin and eosin (H&E) stain and an IHC. Following necropsy of 10 to 13-week-old male Sprague Dawley rats, tissues were collected, trimmed, fixed in neutral buffered formalin (NBF), and placed into the corresponding decalcifying solution. After a pilot study with multiple timepoints for both decalcifying solutions, times were selected for the definitive study. Incisors in the definitive study were decalcified for 72, 96 or 120 hours in Immunocal and 24 hours in Decal Stat, trimmed, processed, embedded in paraffin, and sectioned. The microtomy process and sections were evaluated by histotechnologists. Sections were stained withH&E or an IHC to detect vimentin. Veterinary pathologists used blinded assessment to evaluate staining and tissue quality. The H&E sections from Immunocal timepoints scored higher based on criteria such as cellular morphology. However, tissue quality decreased at 120 hours with Immunocal but was adequate after 24 hours with Decal Stat. For IHC, moderate to excellent expression of vimentin was observed at timepoints for both decalcifiers. Optimal tissue sectioning and histological quality were achieved on incisor sections decalcified for 96 hours with Immunocal and 24 hours with Decal Stat.

Optimization of ovine bone decalcification for increased cellular detail: a parametric study

ABSTRACT There are many published methods of decalcifying bone for paraffin histology; however, the current literature lacks details regarding the processing of ovine tissue. Ovine bone tissue presents challenges, as samples are often denser and larger than other comparative animal models, thus increasing decalcification times. Trifluoroacetic Acid (TFAA) has previously been used to decalcify ovine bone samples for histological analysis. Unfortunately, TFAA is a strong acid and often results in loss of cellular detail, especially in the connected soft tissue. This is generally manifested as over staining with eosin, and a decrease in cellular features which impacts overall histological interpretation. It is well known that leaving tissue in acid for long periods degrades cellular detail; therefore, minimizing decalcification time is critical to accurately determine cellular morphology. Six decalcification solutions (8% TFAA, 20% TFAA, 8% formic acid, 20% formic acid, Formical-4, and XLCal, and three temperatures (room temperature, 30°C, 37°C), were examined to determine their effects on cellular detail in ovine vertebrae and humeral heads. These data clearly indicate that 20% formic acid at 30°C yielded better decalcification rates (2.6 d ± 0.9 d) and cellular detail (none to mild changes) for the vertebrae samples, and 20% formic acid at RT yielded the best cellular detail (none to minimal loss) for humerus samples with a moderate amount of time (6.5 d ± 1.7). These results should establish the optimal demineralization procedures for ovine bone used in scientific studies resulting in improved cellular detail while minimizing decalcification times.

Time is bone — Quantitative comparison of decalcification solvents in human femur samples using dual-X-ray-absorptiometry and computed tomography

IntroductionBone decalcification is a necessary preprocessing step in histological and anatomical studies. Several solutions for decalcification with different claimed times for full decalcification are commercially available. Current literature lacks direct, quantitative measurement of calcium hydrocyapatite degradation during decalcification to compare different solutions. Therefore, the aim of this study was to test the performance of three different decalcification solutions in human bone by direct measurement of calcium hydroxyapatite using dual-X-ray-absorptiometry (DEXA) and volumetric computed tomography (CT). MethodsFour femur slices were acquired from the proximal femur of a 76-year-old body donor. The slices were submerged in formaldehyde (control), EDTA, Osteosoft (Merck, Darmstadt, Germany) and “Rapid Bone Decalcifier” (RBD) (American MasterTech Scientific, Lodi, USA). Consecutive DEXA and CT scans were performed at 2 h, 4 h, 8 h, 11 h, 20 h, 44 h and 77 h after solutions were added. Besides the calcium hydroxyapatite concentration, the bone volume was measured each time. ResultsFastest decline in volume was seen in the RBD probe. Further, RBD was the only solution, being able to fully decalcify the bone slice after 77 h. Although a steady decline in volume and hydroxyapatite concentration was seen for EDTA and Osteosoft as well, both were not able to decalcify the slices. ConclusionOverall, the purely qualititve acquired literature data on bone decalcifiers was verified by our quantitative data for human, cortical-rich bones. Hydrochloric-acid based solutions seem to be preferable in order to rapidly dissolve the calcium hydroxyapatite.

Isolation of DNA from human teeth exposed to different decalcifying solutions for forensic identification: A study

Background: Teeth are selected as source of DNA material mainly due to the high durability found in its structures (enamel, cementum, and dentin), which often succeeds in preserving the integrity of genetic material. Attempts of perpetrators of crime in the destruction of evidence including dead bodies are on the rise. Hence, the use of advanced techniques such as DNA analysis in such a scenario has been the choice. Aim: The purpose of this study was to evaluate the extraction and amplification of DNA from human teeth exposed to different chemicals Nitric Acid at 25%, Formaldehyde at 25 %, and Acetic Acid at 25 %. Objective: The objective of the study was to determine the intactness of DNA using polymerase chain reaction. Materials and Methods: The experimental study was performed with a sample of 15 subjects who underwent tooth extraction from which 5 samples of oral mucosal cells were taken as controls (reference population). The experimental population was divided into three equal parts, which were exposed to different chemical solutions, namely Nitric Acid 25 %, Formaldehyde at 25 %, and Acetic Acid at 25 % compared with the control group (oral mucosal cells [5 samples]). The silica method was used for the extraction of DNA from teeth and the organic method was used for the extraction of DNA from oral mucosa cells. Results: Estimation of DNA quantity and size distribution was done on an Agrose Gel Electrophorosis. From our study we could observe that the teeth that were immersed in 25% Formaldehyde & 25% Acetic Acid were having intact DNA, which we were able to isolate & amplify. There was degradation of DNA tooth which were immersed in 25% Nitric Acid, thus the identification & amplification was not possible. Conclusion: The present study demonstrated that DNA extraction may be limited under exposure to chemical solutions or bodies that undergo intentional postmortem alterations, such as carbonization and dissolution, hamper the degradation of DNA due to high temperature and acid pH. Therefore, testing the collection of DNA from the human teeth in such conditions could contribute significantly to the field of forensic genetics.

Comparison between Conventional Decalcification and a Microwave-Assisted Method in Bone Tissue Affected with Mycetoma.

Mycetoma is a lifelong granulomatous disease of subcutaneous tissues and bones. Histopathology is a substantiated indicative method based on the assumption of a definitive diagnosis of mycetoma. It requires efficient processing of tissues including bone decalcification. The decalcification process must ensure complete removal of calcium and also a proper preservation of tissue and microorganisms' staining ability. Objectives. To compare the conventional method used in decalcification with the microwave method using different decalcification solutions. Different characteristics were tested, including the speed of decalcification and morphological and fungal preservation in bone tissue affected with mycetoma. Materials and Methods. Three decalcification solutions were employed to remove calcium from 50 bone tissue samples affected with mycetoma, including 10% neutral buffered EDTA (pH 7.4), 5% nitric acid, and 5% hydrochloric acid. Conventional and microwave methods were used. Haematoxylin-eosin (HE) stain, Gridley's stain, and Grocott hexamine-silver stain were employed to evaluate the bone and fungi morphologies. Results. The decalcification time of the conventional method compared with the microwave method with 10% EDTA (pH 7.4) took 120 hours and 29 hours, while 5% hydrochloric acid and 5% nitric acid took 8 hours and 3 hours, separately. Also, 10% EDTA is the best decalcifying agent for HE staining and fungal stains. 5% hydrochloric acid and 5% nitric acid can be used for fungal staining. Conclusion. The current study investigated the effects of different decalcifying agents as well as two decalcification procedures on the preservation of the bone structure and fungal staining, which will help to develop suitable protocols for the analyses of the bone tissue affected with mycetoma infection.

Comparison of Methods for the Histological Evaluation of Odontocete Spiral Ganglion Cells.

Simple SummaryHearing is arguably the primary sensory and communication channel for cetaceans. The study of diverse physiological and pathological events involving this system, requires standardized and reliable protocols for processing valuable and scarce samples such as the ears of cetaceans. As part of our research, standardized tissue processing protocols were developed in order to improve our knowledge regarding the anatomy and pathological changes of this sensorial system, including the nervous structures of the ear. The results obtained suggest that ethylenediaminetetraacetic acid (EDTA)-based decalcifying solutions are key for correct evaluation of bony tissue structures such as the ear of cetaceans.Cetaceans greatly depend on their hearing system to perform many vital activities. The spiral ganglion is an essential component of the auditory pathway and can even be associated with injuries caused by anthropogenic noise. However, its anatomical location, characterized by surrounding bony structures, makes the anatomical and anatomopathological study of the spiral ganglion a difficult task. In order to obtain high-quality tissue samples, a perfect balance between decalcification and the preservation of neural components must be achieved. In this study, different methodologies for spiral ganglion sample preparation and preservation were evaluated. Hydrochloric acid had the shortest decalcification time but damaged the tissue extensively. Both formic acid and EDTA decalcification solutions had a longer decalcification time but exhibited better preservation of the neurons. However, improved cell morphology and staining were observed on ears pretreated with EDTA solution. Therefore, we suggest that decalcifying methodologies based on EDTA solutions should be used to obtain the highest quality samples for studying cell morphology and antigenicity in cetacean spiral ganglion neurons.

Functionalized Graphene Oxide Shields Tooth Dentin from Decalcification

This in vitro study assessed the efficacy of functionalized graphene oxide (f-GO) nanocomposites on the decalcification of dentin, because dental caries of the root surface is becoming one of the new problems in aged society. Hydroxyapatite plates (HAP) and dentin slices were coated with f-GO nanocomposites by comparing them to silver diamine fluoride as a positive control, then treated with decalcification solutions such as ethylenediaminetetraacetic acid and citrate at 37°C for 24 h. Scanning electron microscopy (SEM) revealed significant protection of the surface morphology of HAP and dentin. On the other hand, a cariogenic Streptococcus mutans growth was inhibited by f-GO nanocomposites. In addition, cytotoxicity of them to epithelial cells was much less than that of povidone-iodine, which is commonly used for oral disinfectant. We synthesized 5 different f-GO nanocomposites such as GO–silver (Ag), GO-Ag–calcium fluoride (CaF2), GO-CaF2, GO-zinc, and GO–tricalcium phosphate (Ca3(PO4)2). They were standardized by evaluating under SEM, transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetry analysis (TGA), and Raman spectra after being synthesized in an aseptic technique. The abilities of GO-Ag, GO-Ag-CaF2, and GO-CaF2 nanocomposites were most preventive for decalcification. In addition, GO-Ag and GO-Ag-CaF2 almost completely inhibited S. mutans growth. However, they did not exhibit cytotoxicity to epithelial cells except at the highest concentration (0.1 w/v%) of GO-Ag and GO-Ag-CaF2. Furthermore, these f-GO nanocomposites exhibited less or no discoloration of dentin, although commonly used silver diamine fluoride causes discoloration of dentin to black. Thus, these f-GO nanocomposites are useful to protect dental caries on the tooth root that becomes a social problem in aged society.

Efektivitas Larutan Dekalsifikasi pada Os tibia Domba Garut (Ovis aries) (THE EFFECTIVENESS OF DECALCIFYING SOLUTIONS ON THE TIBIAL OF GARUT SHEEP (OVIS ARIES))

Bone is a tissue that has a density of extracellular matrix structures and composed by organic and inorganic components. Decalcification is a stage that plays an important role in bone histology using various types of solutions. The sample used in this study was lateral condyle from tibia of three garut sheeps (Ovis aries) which had been fixed with 10% Neutral Buffered Formalin (NBF) for 24 hours. Sample were cut into pieces ranging from 1 cm x 1 cm x 1 cm in size, the decalcification using three solutions; 10% nitric acid, 10% EDTA (pH 7.4) and 10% EDTA (pH 7.4) + TBD-1®. The aim of this study was to evaluate the effectiveness of three solutions for the decalcification process of lateral tibial condyle of garut sheep. Observation parameter in this study includes: duration of decalcification, sectioning process of ribboning formation, structural integrity and absorption of hematoxylin-eosin (HE) staining. The results shows that 10% EDTA (pH 7.4) solution provides a long duration of decalcification which is ease sectioning process ribboning, the best structural integrity of lateral tibial condyle. In hematoxylin-eosin (HE) staining shows that 10% nitric acid solution does not absorb the optimum color, opposite in 10% EDTA and 10% EDTA + TBD-1® solution, the color intensity between hematoxylin and eosin in the tissue shows the best results. Based on these results, it can be concluded that the 10% EDTA (pH 7.4) is the best decalcification solution for lateral condyle from tibial of garut sheeps.