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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.

Ethylenediaminetetraacetic acid (EDTA), a classically used chelating agent of decalcification, maintains good morphological details, but its slow decalcification limits its wider applications. Many procedures have been reported to accelerate EDTA-based decalcification, involving temperature, concentration, sonication, agitation, vacuum, microwave, or combination. However, these procedures, concentrating on purely tissue-outside physical factors to increase the chemical diffusion, do not enable EDTA to exert its full capacity due to tissue intrinsic chemical resistances around the diffusion passage. The resistances, such as tissue inner lipids and electric charges, impede the penetration of EDTA. We hypothesized that delipidation and shielding electric charges would accelerate EDTA-based penetration and the subsequent decalcification. The hypothesis was verified by the observation of speedy penetration of EDTA with additives of detergents and hypertonic saline, testing on tissue-mimicking gels of collagen and adult mouse bones. Using a 26% EDTA mixture with the additives at 45°C, a conventional 7-day decalcification of adult mouse ankle joints could be completed within 24 h while the tissue morphological structure, antigenicity, enzymes, and DNA were well preserved, and mRNA better retained compared to using 15% EDTA at room temperature. The addition of hypertonic saline and detergents to EDTA decalcification is a simple, rapid, and inexpensive method that doesn't disrupt the current histological workflow. This method is equally or even more effective than the currently most used decalcification methods in preserving the morphological details of tissues. It can be highly beneficial for the related community., (© 2024. The Author(s).)

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.

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., (© 2022 American Academy of Forensic Sciences.)

Bone tissue can be involved by primitive or metastatic tumors and requires a specific processing both at the department of pathology and during multidisciplinary meetings. The development of fine-needle percutaneous biopsies and of molecular techniques in bone tumor pathology requires a specific management. Moreover, decalcification of samples is crucial but can be deleterious if not controlled or not appropriate. The aim of this review is to provide recommendations for management and decalcification of bone tumor samples., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

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.

Bone is the most common site of metastasis in breast carcinoma (BC). Treatments for metastatic BC depend on various factors, including the tumor's estrogen receptor (ER), progesterone receptor (PR), and HER2 status. Bone biopsies require decalcification which may affect the accuracy of ER and PR immunohistochemistry (IHC) and HER2 situ hybridization (FISH) studies. Ethylenediaminetetraacetic acid (EDTA) decalcifying solutions have been theorized to have no significant impact on ER and PR IHC or HER2 FISH analyses. We completed a prospective study of the effect of EDTA decalcification on ER and PR IHC and HER2 FISH in 29 cases of BC. Samples from 29 BC resections were collected and formalin fixed between 12 and 24 h. Control samples were routinely processed, whereas test samples were placed in EDTA for 48 h. ER and PR slides were blinded, randomized, and evaluated. Blinded samples underwent HER2 FISH assays where an average HER2 copy number and HER2/CEP17 ratio were calculated. Paired differences between EDTA and control samples were compared for ER and PR positivity, average HER2 copy number, and HER2/CEP17 ratios using paired-samples t-tests (PST) and Wilcoxon signed-rank test (WSR). PST and WSR tests yielded no significant difference between EDTA and control tissue for ER% (PST: P = 1; WSR: P = 0.916), PR% (PST: P = 0.973; WSR: P = 0.984), HER2 copy number (PST: P = 0.124; WSR: P = 0.103), and HER2/CEP17 ratio (PST: P = 0.25; WSR: P = 0.105). The use of EDTA in bony tissue is therefore a valid decalcification method to ensure accurate assessment of ER and PR IHC and HER2 FISH in metastatic BC., (Copyright © 2021. Published by Elsevier Inc.)

Immunohistochemistry, or immunolabeling, is a key method for the identification of protein expression and localization. Successful detection relies on a low signal-to-noise ratio, which is affected greatly by antibody specificity as well as the staining protocol. Immunohistochemistry in the mouse is challenging, particularly in adult skeletal tissue, due to the need for long decalcification, high autofluorescence and high levels of endogenous peroxidase. Here, we describe a highly sensitive protocol for protein detection in decalcified paraffin-embedded sections from adult mouse skeletal tissue. By using four levels of amplification, this method allows for the identification of even low-abundance proteins.

Cartilage and bone are specialized skeletal tissues composed of unique extracellular matrices. Bone, in particular, has a highly calcified or mineralized matrix that makes microtomy and standard histological studies very challenging. Therefore, methods to appropriately fix and decalcify mineralized skeletal tissues have been developed to allow for paraffin processing and standard microtomy. In this chapter, we will illustrate methods for tissue grossing, fixation, decalcification, paraffin processing, embedding, sectioning, and routine histological staining of demineralized murine skeletal tissues. We will also discuss methods for decalcified frozen sectioning of skeletal tissues with and without the use of a tape-transfer system.

Calcination and decalcification are basic procedures useful to a morphological approach of a biological, composite material like cortical bone. The study was carried out on a whole human femur conserved in liquid (from an educational collection). Cortical fracturing and SEM observation of vascular canals surface collagen texture was used to study bone deproteination at scalar temperatures (400-1,200°C) and acid bone decalcification at crescent time intervals. Heating burned and vaporized the organic matrix with shrinkage of the bone specimens as documented by the weight loss and transverse surface morphometry. SEM showed a pattern of aligned spherulites at 400°C which maintained the collagen fibrils layout (like a mineral cast), followed by a spherulites fusion progression with the temperature increments. At 1200°C a crystalline-like structure of tightly-packed trapezohendron units. XRD analysis supported the SEM morphology displaying the complete Debey rings of hydroxyapatite and spotted Debey rings of withlockite. Surface Ca and P elution was documented after 12 hr of exposition to the acid solution by dissolution of spherulites and the whole canal surface decalcified in depth after 15 days by SEM-EDAX analysis. The periodic pattern of collagen fibrils was still evident up to 15 days of decalcification together with fine granular deposits of a not-collagenic proteic material, while after 30 days no period was observed in the decalcified fibrils. Collagen mineral cast at 400°C calcination. Complete crystalline transformation at 1200°C. Up to 15 days of decalcification fibrils period maintained., (© 2020 Wiley Periodicals, Inc.)

Diagnosis of osteocartilaginous pathologies depends on morphological examination and immunohistochemical and molecular biology analyses. Decalcification is required before tissue processing, but available protocols often lead to altered proteins and nucleic acids, and thus compromise the diagnosis. The objective of this study was to compare the effect of different methods of decalcification on histomolecular analyses required for diagnosis and to recommend an optimal protocol for processing these samples in routine practice. We prospectively submitted 35 tissue samples to different decalcification procedures with hydrochloric acid, formic acid, and EDTA, in short, overnight and long cycles for 1 to >10 cycles. Preservation of protein integrity was examined by immunohistochemistry, and quality of nucleic acids was estimated after extraction (DNA and RNA concentrations, 260/280 ratios, PCR cycle thresholds), analysis of DNA mutations (high-resolution melting) or amplifications (PCR, in situ hybridization), and detection of fusion transcripts (RT-PCR, in situ hybridization). Hydrochloric acid- and long-term formic acid-based decalcification induced false-negative results on immunohistochemistry and molecular analysis. EDTA and short-term formic acid-based decalcification (<5 cycles of 6 h each) did not alter antigenicity and allowed for detection of gene mutations, amplifications or even fusion transcripts. EDTA showed superiority for in situ hybridization techniques. According to these results and our institutional experience, we propose recommendations for decalcification of bone samples, from biopsies to surgical specimens.

Bone containing tissues such as osteochondral joint are resistant to routine tissue processing, therefore require decalcification. This technique causes removal of mineral salts, but in the process may macerate the organic tissue, hence the need for tissue fixation. Such severe processing demands careful antigen retrieval to necessitate optimal staining. The aim of our study was to compare five different antigen retrieval protocols (heat retrieval and protein digestion) following decalcification of rabbit knee joints using two different techniques (20% formic acid and 10% ethylenediamine-tetra acetic acid: EDTA). Osteochondral sections were compared based on time required for decalcification, ease of sectioning, morphological integrity using HE staining and antigen preservation (Collagen type II) using immunohistochemistry. The two decalcification solutions did not impair the tissue morphology and ease of sectioning. Joints processed with formic acid decalcified four times faster than EDTA. Among the five antigen retrieval approaches, maximal collagen II uptake with minimal nonspecific staining was found with protein digestion (pronase and hyaluronidase) in both formic acid and EDTA sections. For osteo-chondral sections, we recommend using 10% EDTA for decalcification and pronase plus hyaluronidase for antigen retrieval if maintaining tissue morphology is crucial, whereas if time is of the essence, 20% FA with pronase plus hyaluronidase is the faster option while still preserving structural integrity. Clin. Anat. 33:343-349, 2020. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Axon degeneration destructs functional connectivity of neural circuits and is one of the common, key pathological features of different neurodegenerative diseases. However, conventional histochemistry methods, which largely rely on tissue sections, have intrinsic limitations in examining the 3D distribution of axonal structures on the whole-tissue level. This technical shortcoming has continuously impeded our in-depth understanding of pathological axon degeneration in many scenarios. To overcome such drawback encountered in the research field, we describe here a general protocol of whole-tissue immunolabeling and 3D fluorescence imaging technique to visualize axon degeneration in the intact, unsectioned mouse tissues. In particular, experimental steps of tissue harvesting, whole-tissue immunolabeling, tissue optical clearing, and 3D fluorescence imaging have been systematically optimized, which makes the protocol effective for assessing integrity of the axonal structures in a variety of tissues. Notably, it has enabled the 3D fluorescence imaging of chemotherapy- or traumatic injury-induced axon degeneration within the bones (e.g., femurs) or bone-containing tissues (e.g., hindpaws), which had previously been inaccessible to conventional histochemistry methods. This protocol is therefore readily compatible with many areas of the research on axon degeneration and is poised to serve the field in future investigations.

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.

Conventional bone decalcification is a time-consuming process and is therefore unsuitable for clinical applications and time-limited research projects. Consequently, we compared the effect of four different decalcification solutions applied at three different temperatures, and assessed the rate of decalcification and the implications on tissue morphology and antigenicity of mouse and rat tibiae. Bones were decalcified with 10% ethylenediaminetetraacetic acid (EDTA), 10% formic acid, 5% hydrochloric acid, and 5% nitric acid at 4C, 25C, and 37C. Decalcification in both species was fastest in nitric acid at 37C and slowest in EDTA at 4C. Histological and immunohistochemical staining confirmed that the conventional protocols of EDTA at 4C and 25C remain the best option regarding the quality of tissue preservation. Whereas formic acid at 4C is a good alternative saving about 90% of the decalcification time, hydrochloric and nitric acids should be avoided particularly in case of rat tibia. By contrast, due to their smaller size, mouse tibiae had shorter decalcification times and tolerated higher temperatures and exposure to acids much better. In conclusion, this study demonstrated that depending on the specific research question and sample size, alternative decalcification methods could be used to decrease the time of decalcification while maintaining histological accuracy.

Fixation and decalcification can alter protein structure in tissues, influencing the efficacy of primary antibodies routinely used in immunohistochemical (IHC) staining. Histologic examination of zebrafish requires both processes, making staining and analysis potentially challenging. Here, we investigated the effects of common fixation and decalcification protocols on IHC staining in zebrafish. We also identified zebrafish-reactive and -specific antibodies for use in research and diagnostics. For several of the antibodies, time spent in Dietrich's fixative containing 2% glacial acetic acid or 3.4% formaldehyde followed by decalcification with ethylenediaminetetraacetic acid (EDTA) significantly impacted IHC staining quality, particularly regarding staining intensity. Protocols utilizing shorter fixation times produced higher-quality stains. In addition, individual markers were variably affected by the type of fixative. Dietrich's fixative significantly reduced staining quality for the "neural" markers: glial fibrillar acidic protein, chromogranin A, S100. A negative time-dependent effect of fixation on staining quality was found for several antibodies: muscle actin (Dietrich's only), cytokeratin AE1/AE3, chromogranin, and S100. Neither decalcification protocol had a statistically significant negative time-dependent effect on staining quality. Based on our results, we suggest shorter fixation and decalcification protocols to best preserve IHC staining quality as well as recommend deliberate selection of the fixative used depending on the protein of interest.

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

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin-fixed and paraffin-embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morsés solution (Group V). Serial, 3-μm-thick sections were obtained and stained with hematoxylin-eosin to calculate the percentage of osteocyte-occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi-quantitative analysis to verify the adjacent muscle-to-bone integrity and preservation of collagen fibres. The highest percentage of osteocyte-occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8-5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p < .001). Group IV (EDTA) had the highest integrity of muscle and collagen type I and III (P < 0.01). Demineralization with 10% acetic acid was the most effective at preserving bone tissue, while 5% EDTA was the best at maintaining collagen and adjacent muscle to bone. In conclusion, nitric acid at 5% showed the most efficient result as it balanced both time and cost as a demineralizing solution., (© 2016 Wiley Periodicals, Inc.)

Bone metastasis from primary cancer sites creates diagnostic and therapeutic challenges. Calcified bone is difficult to biopsy due to tissue hardness and patient discomfort, thus limiting the frequency and availability of bone/bone marrow biopsy material for molecular profiling. In addition, bony tissue must be demineralized (decalcified) prior to histomorphologic analysis. Decalcification processes rely on three main principles: (a) solubility of calcium salts in an acid, such as formic or nitric acid; (b) calcium chelation with ethylenediaminetetraacetic acid (EDTA); or (c) ion-exchange resins in a weak acid. A major roadblock in molecular profiling of bony tissue has been the lack of a suitable demineralization process that preserves histomorphology of calcified and soft tissue elements while also preserving phosphoproteins and nucleic acids. In this chapter, we describe general issues relevant to specimen collection and preservation of osseous tissue for molecular profiling. We provide two protocols: (a) one-step preservation of tissue histomorphology and proteins and posttranslational modifications, with simultaneous decalcification of bony tissue, and (b) ethanol-based tissue processing for TheraLin-fixed bony tissue.

Aim . In routine histopathology, decalcification is an essential step for mineralized tissues. The purpose of this study is to evaluate the effects of different decalcification solutions on the morphological and antigenicity preservation in Sprague Dawley (SD) rat femurs. Materials and Methods . Four different decalcification solutions were employed to remove the mineral substances from rat femurs, including 10% neutral buffered EDTA, 3% nitric acid, 5% nitric acid, and 8% hydrochloric acid/formic acid. Shaking and low temperature were used to process the samples. The stainings of hematoxylin-eosin (HE) and immunohistochemical (IHC) were employed to evaluate the bone morphology and antigenicity. Key Findings . Different decalcification solutions may affect the quality of morphology and the staining of paraffin-embedded sections in pathological examinations. Among four decalcifying solutions, 3% nitric acid is the best decalcifying agent for HE staining. 10% neutral buffered EDTA and 5% nitric acid are the preferred decalcifying agents for IHC staining. Significance . The current study investigated the effects of different decalcifying agents on the preservation of the bone structure and antigenicity, which will help to develop suitable protocols for the analyses of the bony tissue., Competing Interests: The authors declare no conflict of interests regarding the publication of this paper.

Objectives: To determine the feasibility of a rapid method of processing mandible bone margins for intraoperative histopathologic examination and to assess the relative value of fine, coarse, and core specimens in assessing bone margins., Study Design: Prospective histologic controlled study., Setting: A tertiary level academic medical center histopathology laboratory., Subjects and Methods: Multiple bone samples were collected from fresh (<12 hours post-mortem) human cadaveric mandible using a 1) standard 4mm otolaryngologic cutting drill bit 2) diamond drill bit and 3) cutting core biopsy trocar. The specimens were placed in one of three decalcifying solutions (Decal A, Calex, EDTA Decal) from 15 to 75 minutes or control (fixation in 10% formalin). After each designated decalcification time period, specimens were cryosectioned or paraffin embedded and subsequently reviewed by a head and neck surgical pathologist. The specimens were assessed for overall quality, adequacy of decalcification, soft tissue quality, marrow quality, and presence of artifact., Results: Bone margin specimens collected with a 4mm burr and processed with EDTA Decal for 30 minutes yielded the highest quality histopathologic slides compared to the other methods in a similar time frame. The adequacy of decalcification directly impacted the quality of histopathologic assessment., Conclusions: Mandible bone margins can be rapidly and safely prepared and adequately evaluated with only 30 minutes of decalcification. This method may provide acceptable intraoperative assessment of bone margins in patients with tumors which involve or approximate bone. We plan to examine this model in a prospective clinical study of patients with cancer invading mandibular bone., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aim: There are various techniques to study root canal morphology and diaphonization is one of them. There are various methods of decalcification and diaphonization, cited in literature and the main aim of this paper was to give a brief account of the various techniques and share our experience of the technique at a teaching institution in Karachi, Pakistan., Materials and Methods: Diaphonization is one of the oldest methods and is based on decalcification of teeth followed by clearing and dye penetration. The specimen is later studied under microscope without sectioning., Results: After the process of clearing a three-dimensional (3D) structure of the internal canal anatomy was visible with naked eye., Conclusion: This paper entails a detailed historical background as well as the author's technique including percentages of various chemicals used and the timing of immersion of teeth into these agents., Clinical Significance: The read out is simple and can be subjected to interpretation by direct observation under microscope and can be helpful for students undertaking research in not only the discipline of dentistry but also in other fields such as botany and zoology.

In anatomic pathology, the current state encompassing the pre-analytic processes of tissue collection, handling, examination, preparation, processing, and storage are largely uncontrolled, inconsistently performed, and/or not standardized according to the sound scientific data. Pre-analytic defects result in nearly three-quarters of the problems in laboratory diagnostics. This is evident in quality surveys from well-respected institutions that document high miss rates in the required basics of information related to patient and tissue identity, let alone parameters documenting quality aspects related to the surgical specimen and its preservation. This talk will describe the historical approach to tissue processing and identify gaps from worldwide observations in current laboratory practices. It will also offer potential methodological and technological solutions and process improvements that laboratories may consider in serving the ultimate users of pathology information: the clinician and the patient. It illustrates the need for scientifically validated specimen guidelines and a performance based, standardized and documented "chain of custody" of the pre-analytical steps from the patient's body through fixation. For thought leaders and professional standard setters, opportunities for optimizing molecular studies exist in specimen collection, transfer, grossing, fixation, and decalcification protocols. In this evolving era of molecular profiling and personalized therapeutic decision-making, a well-reasoned and coordinated focus on pre-analytic processes that optimizes specimens for subsequent testing will result in: Improved specimen quality for molecular testing Improved accuracy of diagnostic and molecular test results Reduced Turnaroundtimes for same-day diagnosis Enhanced satisfaction of clinicians and patients.

Introduction: The purpose of this study was to study the root canal morphology of South Asian Indian mandibular premolars using a tooth clearing technique., Methods: Two hundred mandibular premolar teeth were collected from different dental schools and clinics in India. After pulp tissue removal and root canal staining with Indian ink, the specimens were decalcified with 5% nitric acid, dehydrated in ethyl alcohol, and subsequently cleared in methyl salicylate., Results: Of the 200 mandibular premolars, 100 were first premolars and 100 were second premolars. Of the first premolars, 94% had a single root, whereas 6% were 2 rooted. Seventy-six percent had a single canal, 22% had 2 canals, and 2% had 3 canals. Eighty-two percent had a single apical foramen, 16% had 2 foramens, and 2% teeth had 3 apical foramens. Eighty percent of teeth had type I, 6% had type II, 10% had type IV, 2% had type V, and 2% teeth had type IX root canal anatomy. Of the 100 second premolars, 92% had a single root, whereas 8% teeth were 2 rooted and fused. Fifty-eight percent of teeth had a single canal, and 42% had two canals. Eighty-eight percent had a single apical foramen, and 12% had 2 foramens. Sixty-six percent had type I, 30% had type II, and 4% had type V root canal anatomy., Conclusions: A high prevalence of 2 canals was noted in the first and second premolars. Also, 20% of first premolars and 34% of second premolars had a root canal anatomy other than type I., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen-antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Analysis of DNA from older samples, such as museum specimen, is a promising approach to studying genetics of populations and ecological processes across several generations. Here, we present a method for extracting high quality nuclear DNA for microsatellite analysis from antlers of red deer (Cervus elaphus). The genotyping of individuals was based on nine microsatellite loci. Because the amount of DNA found in antlers was high, we could reduce the amount of sample and chemicals used and shorten the decalcification time in comparison to other methods. Using these methods, we obtained genotypes from antlers up to 200 years old., (© 2013 Blackwell Publishing Ltd.)

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 present study included six different decalcifying solutions: 10% formal nitric acid, 8% formal nitric acid, 10% formic acid, 8% formic acid, Perenyi's fluid and Ethylene Di-Amine Tetra Acetic Acid. eight samples of posterior mandible of rat were decalcified in each of the decalcifying solutions and subjected to chemical end-point test. Ehrlich's Hematoxylin stain was used., Statistical Analysis Used: One way ANOVA was used for multiple group comparisons and Chi-square test was used for analyzing categorical data. P value of 0.05/less was set for statistical significance., Results: Samples treated with EDTA showed the best overall histological impression and the tissue integrity were well preserved. Formal nitric of both the percentages 10 and 8% gave fairly good cellular detail and were rapid in their action., Conclusion: The final impression led to the proposition that EDTA was indeed the best decalcifying agent available. However, with time constraint, the use of formal nitric acid is advocated.

Objective: To establish a rapid, simple, and economic method to prepare osteoporosis (OP) in vitro model., Methods: Eighty pairs of fresh goat femur were collected from 18-month-old female goats and were randomly divided into 4 groups (20 pairs in each group). The femur was immersed decalcifying solution (18% EDTA) for 1-5 days (group B), 6-10 days (group C), and 11-15 days (group D), while group A had no treatment as control. Four pairs of femur were taken out every day. Quantitative computed tomography was used to scan the medial and lateral femoral condyles, and the bone mineral density (BMD) was calculated. Electronic universal testing machine was used to do three-point bending test and compress and tensile ultimate strenght test, and the mechanical parameters for femur were calculated., Results: With demineralized time passing, BMD of the medial and lateral femoral condyles were downtrend in groups A, B, C, and D, showing significant differences among 4 groups (P < 0.05); BMD of the lateral femoral condyle was significantly higher than that of the medial femoral condyle in each group (P < 0.05). The three-point bending test showed that broken load, ultimate strength, and elastic modulus of groups A and B were significantly higher than those of groups C and D (P < 0.05); but no significant difference was found between groups A and B, and between groups C and D (P > 0.05). Compress and tensile ultimate strength test showed that the compress and tensile ultimate strengths were significantly higher in group A than in groups C and D (P < 0.05), and in group B than in group D (P < 0.05), but no significant difference was found between groups A and B, between groups B and C, and between groups C and D (P > 0.05)., Conclusion: The 18% EDTA immersing for 6-15 days is a fast, simple, economical method to prepare an OP in vitro model of goat femur.

Hypothesis: Decalcification of cochlear samples in Morse's solution after methacarn fixation provides greater RNA quantification and morphologic preservation of cochlear structures as compared with EDTA and formic acid decalcifying solutions after methacarn fixation., Background: A variety of fixatives and decalcifying agents can fragment or chemically alter RNA in samples inhibiting their isolation and quantification. Morphologic alterations can also be observed in light microscopy analyses. The cochlea is embedded in the bone; hence, fixation and decalcification steps are mandatory to obtain histologic sections and preserve the cochlea for morphologic evaluation., Methods: Cochlear samples obtained in a RNase-free environment were processed in 4 combinations of decalcifying agents in combination with methacarn fixation. Samples in Protocols 1, 2, and 3 were fixed in methacarn for 4 hours at 4°C, followed by decalcification at 4°C with Morse's solution, 10% ethylenediaminetetraacetic acid, and 5% formic acid solution, respectively. Samples processed with protocol 4 were decalcified in Morse's solution at 4°C followed by fixation for 4 hours at 4°C. Real-time PCR analysis was performed on total RNA extracted. Histology sections were evaluated for morphology preservation of cochlear structures., Results: RNA was isolated in all samples. Relative expression levels were greatest with Protocol 1 and lowest with Protocol 3. Morphology preservation was adequate with Protocols 1, 2, and 3., Conclusion: Of the 4 protocols evaluated, methacarn fixation followed by decalcification in Morse's solution provided the greatest genetic expression levels as well as the best tissue morphology preservation in the cochlea.

The aim of this study was to determine the possibility of immunohistochemical demonstration of various tissue antigens in paraffin sections of zinc-ethanol-formaldehyde-fixed spinal cord, surrounded by the vertebral tissues and adjacent muscles, after decalcification in formic acid, in adult (n = 5) and newborn (n = 2) Wistar rats. In the present work, the antibodies used were against: glial fibrillary acidic protein, neurofilaments, neuronal specific nuclear protein, enzyme tyrosine hydroxylase, protein of synaptic vesicles--synaptophysin and the intermediate filament protein--vimentin. Combination of fixation in zinc-ethanol-formaldehyde and decalcification in 21% formic acid solution was shown to provide an optimal preservation of the antigens studied.

The paper describes a decalcification procedure that may be used in any histology laboratory handling bone samples for general and special purposes. It discusses the advantages and disadvantages of the major types of decalcifying agents, the principles of their choice for different types of objects, and the methods for determining the endpoint of decalcification.

This study was performed to elucidate the relationship between dental caries and the levels of Sr and Ca eluted from enamel, and to examine whether these elements are useful as factors to assess caries risk. The available 103 (Sr) and 108 (Ca) samples were obtained among 111 collected deciduous teeth. The healthy regions of enamel were decalcified in artificial saliva at pH 6.2 and 5.5. The eluted levels of these elements from enamel were determined using atomic absorption spectrophotometry. Sr and Ca levels were not affected by the sex nor tooth type. Sr levels of the caries-experienced tooth (CE) group were 2.6-fold (pH 6.2) and 2.2-fold (pH 5.5) higher than those of the sound tooth (ST) group, respectively. Furthermore, the Sr levels were significantly higher in the teeth with treated than in those with untreated caries. Only at pH 6.2 was a significant difference found in Ca levels between the ST and CE groups. In the ST group, at pH 5.5, both the Sr and Ca levels significantly increased when the children had six or more carious teeth. The Sr and Ca elution levels were significantly inhibited in the teeth receiving fluoride application every 3 or 4 months compared to those that were not. These findings indicate that Sr can be an indicator of the acid resistance of teeth, and a useful factor to assess future caries risk.

Mitral valve replacement (MVR) in the presence of the extensive calcification of the mitral annulus is a technical challenge. The heavily calcified annulus can cause great difficulty in the insertion of a prosthetic valve and periprosthetic leakage later on. Vigorous annular decalcification may cause circumflex coronary artery injury, atrioventricular rupture and thromboembolic events. We herein describe a surgical technique for MVR in such cases while focusing on partial decalcification of the posterior mitral annulus and its reinforcement and buttressing with the transferred anterior mitral leaflet (AML). At the same time, the transferred AML supports the posterior annular region and maintains ventricular-annular continuity, thus preserving the left ventricular function.

Objective: To evaluate the tensile mechanical characteristics of decalcified cortical bone matrix with different thicknesses so as to provide an experimental basis for the scaffold of tissue engineering., Methods: Decalcified cortical bone matrix was prepared from fresh bovine tibia with rapid decalcification techniques. Its physical characteristics including colour, texture, and so on, were observed. Then the decalcified rate was calculated. Decalcified cortical bone matrices were radially cut into slices with different thicknesses along longitudinal axis and divided into 4 groups: group A (100-300 microm), group B (300-500 microm), group C (500-700 microm), and group D (700-1000 microm). Then the slice specimens of each group were characterized with tensile test and histological examination., Results: General observation showed that decalcified cortical bone matrix with hydrogen peroxide treatment was ivory white with good elasticity and flexibility. The decalcified rate was 97.6%. The tensile strength and elastic modulus of groups B, C, and D were significantly higher than those of group A (P < 0.05); there was no significant difference among groups B, C, and D (P > 0.05). The stiffness in 4 groups increased gradually with the increasing thickness, it was significantly lower in group A than those in groups B, C, and D (P < 0.05), and in groups B and C than that in group D (P < 0.05). While there was no significant difference in ultimate strain within 4 groups (P > 0.05). Histologically, intact osteon was observed in every group, with an average maximum diameter of 182 microm (range, 102-325 microm)., Conclusion: The mechanical properties of decalcified cortical bone matrix might depend on the integrity of the osteons. Slices with thickness of 300 microm or more could maintain similar mechanical properties when decalcified cortical bone matrix is used as a scaffold for tissue engineering.

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Objective: To investigate the effects of 1% hydrochloric acid and 10% nitric acid on immunohistochemical staining for several antigens., Methods: Fifteen cases of focally calcified soft tumors including three cases of thyroid papillary carcinoma, three of breast invasive ductal carcinoma, three of prostate cancer, three of colon adenocarcincoma and three of normal lymph nodes, were selected in this study, and so were nine cases of bone and bone marrow tissue from biopsy and autopsy. Tissue samples were divided into three groups. One group was each soaked into 10% nitric acid for 24, 42 and 48 h, another group was each incubated in 1% hydrochloric acid for 1, 2 and 3 h. The other group which was not treated with acid was regarded as control. Then immunohistochemical staining was conducted in these samples for antigens as follows: ER, PR and Ki-67 in breast invasive ductal carcinoma; TTF-1 in thyroid papillary carcinoma; 34βE12 and P504s in prostate cancer; AE1/AE3, P504s and Ki-67 in colon adenocarcincoma; CD3, CD20 and Ki-67 in lymph nodes; CD3,CD20,CD68,CD235a,MPO,Ki-67,AE1/AE3 and TTF-1 in bone tissue., Results: After the tissues decalcificated in both acid solutions for some time, the numbers of positive cells decreased and the intensity of immunostaining became weaker, which was proportionate to time. Bone tissue was less influenced than calcified soft tissue. Different antigens showed different sensitivity to the same acid treatment. TTF-1 and Ki-67 were the two most vulnerable antigens among them; while ER, CD3 and CD20 were the least influenced. The effects of decalcification on immunostaining of one antigen were the same in different tissues., Conclusion: In most cases, both hydrochloric acid and nitric acid decalcification can lower the sensitivity of immunohistochemical staining, but have different effects on different antigens. It is better to use 10% nitric acid for decalcification of relatively large bone tissue and to use 1% hydrochloric acid for bone marrow tissues and bone tissues containing much more soft tissue.

Histological analysis of bone encased tissue is severely hampered by technical difficulties associated with sectioning calcified tissue. Cryosectioning of bone is possible but requires significant technical adaptation and expensive materials and is often time-consuming. Some decalcifying reagents in common use result in successful cryosectioning in less time but the integrity of the soft tissue of the spinal column is often compromised during processing. This can result in significant loss of cellular detail. In order to find a method that would allow cryosectioning of the bone without loss of structural integrity of the underlying soft tissue we assessed the efficacy of four different decalcifying reagents with respect to their effects on the cellular structure of the myelin of the grey and white matter of the spinal cord. The antigenic integrity of the spinal cord white matter was evaluated using tissue structural integrity and quality of myelin basic protein immunostaining. The result of this research shows that 6% TCA not only decalcifies intact spinal column suitably for cryosectioning but does so without compromising the antigenic integrity of the tissue. The ease of application, speed of processing and a favorable cost-effective profile were secondary benefits noted with the use of the 6% TCA decalcifying solution. The ability to utilize a decalcifying solution that allows for both histomorphometry and immunohistochemistry in the same spinal column segment represents a novel technique that will provide new insights into pathophysiological aspects and therapeutic approaches ispinal cord damage or disease., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images and allows for techniques such as fluorochrome assessment and histomorphometry. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Objective: To explain the development of a new personal technique to study the spiral ligament and stria vascularis in Guinea pig cochleae by obtaining sample tissue without decalcification and to assess its validity for electron microscopy analysis., Material and Method: Samples were taken from five female Guinea pigs weighing 200-250 g and were fixed in glutaraldehyde and paraformaldehyde for analysis of the spiral ligament and stria vascularis ultrastructure by transmission electron microscopy., Results: All of the ultrastructure components in the spiral ligament and stria vascularis could be examined without the need for decalcification., Conclusions: Our method to obtain and analyze samples of cochlea side wall is valid, easy and faster.

Aim: Smear layer removal and collagen fiber exposure may improve periodontal treatment and regeneration. This in vitro study assessed smear layer removal and collagen fiber exposure after tetracycline hydrochloride (TTC) application on root surfaces using scanning electron microscopy (SEM)., Methods and Materials: Root cementum was removed with diamond burs followed by scaling and root planning. Four hundred fifty samples were divided into ten groups: a control (saline application) and nine different TTC concentrations were applied at doses of 10, 25, 50, 75, 100, 125, 150, 200, and 250 mg/ml. The TTC application was performed in all groups in three different ways (passive, brushing, and burnishing) and at three different periods of conditioning (1, 2, and 3 minutes). A previously trained, calibrated, and blind examiner evaluated photomicrographs of the samples using Sampaio's index (2005). Statistical analysis was performed using the Kruskal-Wallis' and Dunn's tests., Results: The concentrations of 50 mg/mL and 75 mg/mL applied by burnishing were the most effective in smear layer removal and collagen fiber exposure. Both the passive mode of application (p=0.0001) and 1 minute period of application (p=0.002) were the least effective., Conclusions: The concentrations of 50 mg/mL and 75 mg/mL applied by burnishing during 2 or 3 minutes were the most effective., Clinical Significance: These parameters may be applied in periodontal procedures involving TTC root conditioning to optimize results.

Screws, clamps and other spinal instrumentation materials are tested using healthy animal and healthy human vertebrae, but the application of similar tests to an osteoporotic vertebra is generally neglected because of high costs and limited availability of high quality and consistent osteoporotic vertebrae. The objective of this study is to develop an in-vitro method to decrease the mineral content of an animal vertebra utilizing decalcifying chemical agents that alters the bone mineral density and some biomechanical properties to such an extent that they biomechanically mimic the osteoporotic spine. This study was performed on 24 fresh calf lumbar vertebrae. Twelve out of these 24 vertebrae were demineralized and the others served as control. A hole was opened in the pedicles of each vertebrae and the bone mineral density was measured. Each vertebra was then placed into a beher-glass filled with hydrochloric acid decalcifier solution. The decalcifier solution was introduced through the holes in the pedicles with an infusion pump. The vertebrae were then subjected to DEXA to measure post process BMD. Pedicle screws were introduced into both pedicles of each vertebrae and pullout testing was performed at a rate of 5 mm/min. The difference of BMD measurements between pre- and post-demineralizing process were also statistically significant (p < 0.001). The difference of pullout loads between pre- and post-demineralizing process were also statistically significant (p < 0.001). The acid demineralizing process may be useful for producing a vertebra that has some biomechanical properties that are consistent with osteopenia or osteoporosis in humans.

Immunohistochemistry of undecalcified bone sections embedded in methyl methacrylate (MMA) is not commonly employed because of potential destruction of tissue antigenicity by highly exothermic polymerization. The aim of the present study was to describe a new technique in which a quick decalcification of bone sections embedded in MMA improves the results for immunohistochemistry. The quality of interleukin 1alpha (IL-1alpha) immunostaining according to the present method was better than the conventional one. Immunostaining for osteoprotegerin (OPG) and the receptor activator of NF-kappaB ligand (RANKL) in bone sections of chronic kidney disease patients with mineral bone disorders (CKD-MBD) was stronger than in controls (postmortem healthy subjects). The present study suggested that this method is easy, fast, and effective to perform both histomorphometry and immunohistochemistry in the same bone fragment, yielding new insights into pathophysiological aspects and therapeutic approaches in bone disease.

Objective: To study the methods of decalcification for making united slices of tooth and affiliated periodontic tissues., Methods: Twenty-one samples containing dog molars and affiliated periodontic tissues were divided into seven mean groups. The pH value of solution, time of decalcification, weight and volume of samples, and content of decalcified calcium were detected. The slices were observed by HE, specific, and immunohistochemical stain., Results: The velocity of decalcification increased with decrease of solution pH. The weight of samples lightened by 37.61%, the volume reduced by 25.97% on average, and calcium decalcified was 174.49 mg per gram humid samples. The EDTA decalcification was slowest, but it was best. Decalcification was fast in Plank-Rycho solution while the section was worst, and faster in the formyl solution containing aluminium chloride than in EDTA, and the section was better., Conclusions: The 50% formyl solution containing aluminium chloride is an ideal decalcifying solution.

Rodent arthritis models are tools to study joint destruction. This chapter describes methods to prepare and analyze histological sections from inflamed paws. Protocols to create decalcified paraffin-embedded as well as undecalcified plastic-embedded sections are provided. Strengths and limitations of these two histological techniques are described. Major staining techniques on hard tissue sections focusing on the evaluation of joint destruction are indicated. Finally, evaluation of arthritic paw sections by histomorphometry, a semiautomated computerized method to measure areas of interest, is described.

2H quadrupolar splitting of deuterated water molecules is a sensitive measure of the order and density of the collagen fibers in articular cartilage. In the calcified zone, near the bone, two pairs of quadrupolar split satellites were previously observed. To examine whether the large splitting observed originates from the presence of calcium ions and hydroxyapatite, one-dimensional 2H single and double quantum filtered spectroscopic imaging were performed on articular cartilage-bone plugs before and after decalcification. After decalcification, the magnitude splitting of the two pairs of satellites did not change and orientation dependency was kept. However, the intensity of the large splitting was greatly enhanced. According to these results the two pairs of satellites do not stem from the presence of calcium ions and hydroxyapatite but originate from the presence of two groups of collagen fibers with different degrees of hydration. The enhanced intensity of the large splitting is attributed to an increased amount of water molecules that fill the void, resulting from the removal of hydroxyapatite, which resides near the fibers responsible for the large splitting. The quadrupolar splitting observed in the trabecular bone was not orientation-dependent, indicating a random orientation of the collagen fibers in that tissue.

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.

A method to obtain thin histological sections (4-5 microm thick) from teeth, using a conventional microtome, is described for incisors, molars and canines from Spanish red deer (Cervus elaphus hispanicus). Decalcifying solutions of formic acid and nitric acid at different concentrations were ineffective. This was not the case for a solution of 14% hypochloric acid and polyvinylpyrrolidone (TBD-1(R) of Shandon). However, the time needed to complete decalcification was longer than the time estimated by other authors. Incisors required between 4 and 6 days, molars between 7 and 15 days, and canines between 8 and 12 days. The preparation of histological thin-sections from teeth with a conventional microtome, required some training or prior specialization, because most of defective or lost material occurred during the preliminary stage of the study. However this technique is inexpensive enough to be implemented in any histological laboratory. The preparations obtained are suitable for growth mark observation in dentine and in cementum for dating studies. Nevertheless, they are not suitable for enamel change studies, because the enamel is completely destroyed during the decalcifying process.

Electrolytic decalcification is a very fast and effective method for removing calcium compounds from bones with minimum damage to tissues. Changes of dimension of tissues in histological sections prepared from specimens decalcified by immersion in a formic acid solution and sections prepared from specimens treated in an electrolytic decalcifier were studied. Heads of mouse foetuses were cut in half, decalcified by one of the above-mentioned methods and embedded in histowax. Dimensional changes of skin, tongue and nasal epithelia in histological sections were evaluated by t-test. Significant shrinking and other unwanted effects of decalcification, such as acidophilia of nuclei, were found in objects decalcified by both methods. No significant differences in the effects of the two methods on tissue dimensions were demonstrated. It is concluded that both decalcification methods are equivalent from the qualitative point of view.