Comparison of various decalcifying agents to evaluate their efficacy

Background: Bone is a biological complex structure primarily comprising collagen and minerals. It is important to demineralize these mineralized tissues to remove their calcium apatite crystals for analysing the sub-cellular, cellular, and fibrillar architecture. Six demineralizing agents’ efficacy was examined by assessing their duration, ease of handling tissue, staining, and histological criteria. The present study aimed to evaluate six commonly used demineralizing agents to identify the best decalcifying agent. Methods: Twenty resected hard tissue specimens (1 cm × 1 cm x 1 cm) from the archives were used in the study. These segments were decalcified by solutions namely 10% nitric acid, 10% formic acid, 14% ethylene di amine tetra acetic acid (EDTA), a mixture of formic acid and hydrochloric acid (formic + HCL) 4% each, and a mixture of formic acid and nitric acid 4% each (formic + HNO3), 10% formal nitric acid further subjected to radiographic endpoint test. Results: The present study confirmed the fact that samples treated with EDTA showed the best overall impression in terms of tissue integrity and histology followed by 10% formal nitric acid which gave fairly good cellular details and was also rapid in the action. Conclusions: Based on the present study findings, we suggest that 10% formal nitric acid is the better decalcifying agent available, considering time and tissue integrity as two main factors.

Study on injectable calcium sulfate bone cement rich in bioactivity / 中华骨科杂志

Objective:To explore the effect of decalcified bone matrix (DBM) rich in biological activity on surgical-grade medical calcium sulfate, and to observe the change of different content of DBM on the physical and chemical properties of calcium sulfate, which provide theoretical basis for the preparation of calcium sulfate bone cement with osteogenic and injectable properties.Methods:DBM with weight content of 0, 5%, 10%, 20%, 30%, 40% was fully mixed with CSH. Dissolve 0.3 g of methyl cellulose in 10 ml of deionized water to prepare a 3% methyl cellulose solution. Methylcellulose solution was added according to the liquid-solid ratio of 0.4. The mixture was evenly stirred to form slurry, then the degradation rate, compressive strength, setting time and and pH value of calcium sulfate in vitrowas measured.Results:The initial setting time and final setting time of calcium sulfate were 4.96±0.20 and 5.83±0.12 min respectively. With the increase of DBM content, the initial setting time and final setting time increased significantly ( F=49.275, P<0.05; F=124.859, P<0.05). The compressive strength of pure calcium sulfate is 23.33±6.35 MPa; when the content is 40%, the compressive strength is only 3.33 MPa. With the increase of DBM content, the compressive strength first increased and then decreased; the content of 5%, 10%, 20% DBM had little effect on the compressive strength ( P>0.05), while the compressive strength of 30% and 40% groups decreased significantly ( t=3.259, P<0.05). DBM with different contents can significantly change the degradation rate of calcium sulfate complex. When the content of DBM is 30% and 40%, the complete degradation time in vivo is only 10 d, while the degradation rate of calcium sulfate is 63% in 30 d. At any time point in vitro degradation, DBM had no significant effect on the pH value of calcium sulfate complex culture medium, and the change law was consistent with that of pure calcium sulfate. Conclusion:With the increase of DBM content, the degradation rate is gradually accelerated, the compressive strength is reduced, and the setting time is prolonged, which is not conducive to the preparation of injectable calcium sulfate cement.

Ultrasonic Decalcification for Mitral Stenosis with Mitral Annular Calcification : a Case Report / 日本心臓血管外科学会雑誌

Mitral valve surgeries for cases with mitral annular calcification (MAC) are challenging because of the operative complications. For a case of MS with MAC, we achieved mitral valve plasty by ultrasonic decalcification alone. An 82-year-old male with edema and dyspnea was diagnosed with AS and MS with MAC. MAC was so severe that MVR was challenging. There were calcifications at the anterior commissure and the anterior mitral leaflet (AML), and removal of them was expected to improve the valve function. Therefore, anterior commissurotomy and ultrasonic decalcification of the anterior commissural annulus was performed using cavitron ultrasonic surgical aspiration (CUSA). Following the resection of the aortic valve, we carried out decalcification of the AML through the aortic valve orifice. After AVR, a trans-esophageal echocardiogram showed MS was ameliorated. Two years after surgery, recurrence of MS was not recognized. Some mitral cases with MAC can be treated by only decalcification to avoid risky valve replacement.

Improvement program on pretreatment of acid decalcified tissue in hematoxylin-eosin staining / 华西口腔医学杂志

OBJECTIVE@#To explore the treatment conditions of acid decalcified specimens and improve the poor quality of sections and unclear structure of hematoxylin-eosin (HE) staining caused by the change in pH in tooth and hard tissue after acid decalcification.@*METHODS@#A total of 20 cases of oral pathological specimens that contain hard tissues were decalcified and treated with routine treatment, concentrated ammonia water immersion treatment, and saturated lithium carbonate solution immersion treatment. The quality and HE staining effects of hard tissue sections treated with different methods were compared.@*RESULTS@#Compared with routine treatment, lithium carbonate saturated solution treatment showed complete sections. Hematoxylin is strongly stained, the nucleus is clear, and the cytoplasm is bright.@*CONCLUSIONS@#Soaking acid decalcified specimens in lithium carbonate saturated solution before embedding in dehydration can neutralize the acidic environment of the tissue. The quality of sections and HE staining effect are improved and are suitable for the pretreatment of acid decalcified tissue samples of oral pathology.

Sequential imaging of a case of choroidal osteoma using swept-source OCT and optical coherence tomography angiography: A 4-year follow-up study

A 33-year-old gentleman was presented with metamorphopsia in the left eye due to choroidal osteoma (CO) complicated by choroidal neovascular membrane (CNVM). Optical coherence tomography angiography (OCTA) proved to be a valuable, noninvasive tool in monitoring treatment response of CNVM. The tumor subsequently underwent decalcification over a period of 4 years. In addition, SS-OCT scans were instrumental in documenting the natural course of the tumor and focal choroidal excavations (FCE), which were found in correspondence with tumor decalcification. Close follow-up is warranted in FCE, secondary to decalcification of CO, as CNVM has been documented to occur on the slope or bottom of eyes with FCE.

Decalcification of Anterior Mitral Valve Leaflet to Repair Moderate Nonrheumatic Mitral Valve Stenosis with Severe Aortic Valve Stenosis / 日本心臓血管外科学会雑誌

A 78-year-old woman was referred to our hospital because of progressive exertional dyspnea due to nonrheumatic severe aortic valve stenosis and moderate mitral valve stenosis with mitral annular calcification. We subsequently performed aortic valve replacement and mitral anterior leaflet decalcification. During surgery, we found that the cause of mitral valve stenosis was calcification of A2 aortic curtain-medial trigon through aortic valve annulus and resected calcification with SONOPET. The postoperative echocardiography revealed good mitral valve motion with mild mitral valve stenosis.

Comparative study of three different decalcifying solutions in bone metastasis specimens with breast cancer / 中华病理学杂志

Objective@#To investigate the optimal strategy for immunohistochemical (IHC) staining in bone metastasis specimens from breast cancer.@*Methods@#Twenty-eight bone metastases specimens from breast cancers were divided into three groups and subjected to different decalcifying agents (group A-10% nitrate, group B-EDTA decalcification, and group C-imported decalcifying solution RapidCal). The effects of those on HE and IHC staining for Ki-67, ER, PR, GATA3, RANK, RANKL, HER2 and HER2 FISH results were assessed.@*Results@#There were no significant differences among three groups in HE morphology and IHC staining. Antigen content in the RapidCal group were all intact; the EDTA group showed a similar staining rate, which was better than the nitrate group (P<0.05). Nitrate group showed marked reduction in nuclear Ki-67 staining, but the loss of cytoplasmic antigens (RANK, RANKL) was less than cell membrane antigen (HER2). For FISH, the RapidCal group and EDTA group showed same results, concordant with IHC staining results. The expression of HER2 protein in the nitric acid group was significantly decreased and chromosome 17 labelling was lost (P<0.05).@*Conclusions@#RapidCal treated bone metastases specimens from breast cancer show excellent sample quality in morphological, IHC and FISH results compared with traditional decalcifying agents. Owing to the longer time of EDTA decalcification, the new decalcifying agent RapidCal plays an important role in quality control and clinical application.

A Case Report of Femoral Endarterectomy and Decalcification Using the Cavitron Ultrasonic Surgical Aspirator / 日本心臓血管外科学会雑誌

<p>A 69-year-old male complained of intermittent claudication of the right leg. Computed tomography revealed a right femoral artery stenosis with severe calcification and intimal thickening extending to the superficial and deep femoral arteries. Femoral endarterectomy and decalcification was carried out using the Cavitron Ultrasonic Surgical Aspirator (CUSA). All arteries were repaired by an ePTFE Y-shaped patch. Postoperative CT showed no stenosis and progressive calcification of the common, superficial and deep femoral arteries 2 years after surgery.</p>

Decalcification: A Simpler and Better Alternative.

Background: Decalcification of calcified tissues plays an important part in histological techniques. However, as it often takes a long time and some procedures decrease the staining qualities of the specimen, many attempts have been made to find methods for accelerating this procedure and ensuring good staining properties. One of the factors that regulates decalcification is temperature. Controlled increase of temperature yields decalcification at a faster rate and also retains the basic molecular arrangement. The aim of the study is to formulate a simpler and better alternative for conventional decalcification. Methods: Thirty freshly extracted periodontally compromised molar teeth without evidence of dental caries were used for decalcification in three groups. In each group 10 teeth were used. Group A: 5% HNO3 was used. Group B: 10% HNO3 and 10% formalin was used. Group C: 10% HNO3 and 20% formalin was used. A constant temperature of 55⁰C was maintained. Complete decalcification was checked using X-ray method. The teeth were sent for routine processing and stained using Haematoxylin and Eosin. Results: Decalcified teeth of Group C containing 10 % HNO3 and 20% formalin proved to be advantageous completing decalcification faster among the 3 groups while maintaining good tissue details. Conclusion: In the present study, it was observed that, regardless of the employed fixative solution, preservation of pulp architecture was best, when a combination of 10% HNO3 and 20% formalin was used as a decalcifying agent.

An Experimental Study for Minimum Level of Decalcification to Detect the Osteolytic Bone Metastasis of Long Bone on Plain Radiography

BACKGROUND: In 1951, Ardran reported that metastatic bone lesions could be detectable on plain radiography with 30% to 50% of decalcification. Authors performed experimental study for minimum level of decalcification to detect the osteolytic bone metastasis of long bone with recent technique of radiographs. METHODS: One pair of fibula and humerus from two cadavers was cut into specimen 1 inch in length. Distal half of specimen was dipped into hydrochloride (HCl) with 15 min interval. All 16 specimens were checked by film-type radiography (FR), computed radiography (CR), digital radiography (DR). To exclude inter-observer's variance, 3 radiologists evaluated images. Calcium amount before and after decalcification was measured and expressed in percentage of decalcification. RESULTS: Osteolytic changes were detectable with 11% to 16% of decalcification for fibula and 3% to 8% for humerus on plain radiography with FR, CR, and DR. CONCLUSIONS: Our study showed that minimum of 3% and maximum of 16% of decalcification is necessary when osteolytic metastatic bone lesions of long bone could be detected on plain radiography.

Proposal of an Appropriate Decalcification Method of Bone Marrow Biopsy Specimens in the Era of Expanding Genetic Molecular Study

BACKGROUND: The conventional method for decalcification of bone specimens uses hydrochloric acid (HCl) and is notorious for damaging cellular RNA, DNA, and proteins, thus complicating molecular and immunohistochemical analyses. A method that can effectively decalcify while preserving genetic material is necessary. METHODS: Pairs of bilateral bone marrow biopsies sampled from 53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl. Pairs of right and left bone marrow biopsy samples harvested from 28 cases were allocated into the EDTA versus HCl comparison group, and 25 cases to the RDO versus HCl comparison group. The decalcification protocols were compared with regards to histomorphology, immunohistochemistry, and molecular analysis. For molecular analysis, we randomly selected 5 cases from the EDTA versus HCl and RDO versus HCl groups. RESULTS: The decalcification time for appropriate histomorphologic analysis was the longest in the EDTA method and the shortest in the RDO method. EDTA was superior to RDO or HCl in DNA yield and integrity, assessed via DNA extraction, polymerase chain reaction, and silver in situ hybridization using DNA probes. The EDTA method maintained intact nuclear protein staining on immunohistochemistry, while the HCl method produced poor quality images. Staining after the RDO method had equivocal results. RNA in situ hybridization using kappa and lambda RNA probes measured RNA integrity; the EDTA and RDO method had the best quality, followed by HCl. CONCLUSIONS: The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.

Comparison of micro-computerized tomography and cone-beam computerized tomography in the detection of accessory canals in primary molars

PURPOSE: This study was performed to compare the accuracy of micro-computed tomography (CT) and cone-beam computed tomography (CBCT) in detecting accessory canals in primary molars. MATERIALS AND METHODS: Forty-one extracted human primary first and second molars were embedded in wax blocks and scanned using micro-CT and CBCT. After the images were taken, the samples were processed using a clearing technique and examined under a stereomicroscope in order to establish the gold standard for this study. The specimens were classified into three groups: maxillary molars, mandibular molars with three canals, and mandibular molars with four canals. Differences between the gold standard and the observations made using the imaging methods were calculated using Spearman's rho correlation coefficient test. RESULTS: The presence of accessory canals in micro-CT images of maxillary and mandibular root canals showed a statistically significant correlation with the stereomicroscopic images used as a gold standard. No statistically significant correlation was found between the CBCT findings and the stereomicroscopic images. CONCLUSION: Although micro-CT is not suitable for clinical use, it provides more detailed information about minor anatomical structures. However, CBCT is convenient for clinical use but may not be capable of adequately analyzing the internal anatomy of primary teeth.

A comparative study of various decalcification techniques.

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.

Fluoride penetration from three orthodontic adhesives: an experimental study / 대한치과교정학회지

OBJECTIVE: To examine the prophylactic potential of 3 orthodontic bonding adhesives: Fuji Ortho SC, Illuminate, and Resilience. METHODS: Thirty-six Wistar Wag rats were randomly divided into 4 groups consisting of 9 rats each. One of the groups received no treatment and was used as a control. In the other groups, individual bands coated with one of the 3 adhesives were cemented to the lower incisors. Enamel samples were obtained after 6 and 12 weeks and analyzed using scanning electron microscopy in combination with energy dispersive spectrometry. RESULTS: Six weeks after band cementation, no fluoride was found in the enamel of the lower incisors. After 12 weeks, there was no fluoride in the enamel of teeth coated with the Resilience composite. However, in the case of the Illuminate composite and the resin-modified glass ionomer Fuji Ortho SC cement, the depth of fluoride penetration reached 2 microm and 4.8 - 5.7 microm, respectively. CONCLUSIONS: Fluoride ions from orthodontic adhesives can be incorporated into the surface layer of the enamel. Orthodontists may apply orthodontic adhesives, such as the Fuji Ortho SC, to reduce the occurrence of caries during orthodontic treatment with fixed appliances.

Orthodontic bonding to acid- or laser-etched prebleached enamel / 대한치과교정학회지

OBJECTIVE: Bonding forces of brackets to enamel surfaces may be affected by the procedures used for bleaching and enamel etching. The aim of this study was to investigate the bonding strength of orthodontic brackets to laser-etched surfaces of bleached teeth. METHODS: In a nonbleached control group, acid etching (group A) or Er:YAG laser application (group B) was performed prior to bracket bonding (n = 13 in each group). Similar surface treatments were performed at 1 day (groups C and D; n = 13 in each subgroup) or at 3 weeks (groups E and F; n = 13 in each subgroup) after 38% hydrogen peroxide bleaching in another set of teeth. The specimens were debonded after thermocycling. RESULTS: Laser etching of bleached teeth resulted in clinically unacceptable low bonding strength. In the case of acid-etched teeth, waiting for 3 weeks before attachment of brackets to the bleached surfaces resulted in similar, but not identical, bond strength values as those obtained with nonbleached surfaces. However, in the laser-etched groups, the bonding strength after 3 weeks was the same as that for the nonbleached group. CONCLUSIONS: When teeth bleached with 38% hydrogen peroxide are meant to be bonded immediately, acid etching is preferable.

Effects of demineralizaton-inhibition procedures on the bond strength of brackets bonded to demineralized enamel surface / 대한치과교정학회지

OBJECTIVE: To study and compare the effects of different demineralization-inhibition methods on the shear bond strength (SBS) and fracture mode of an adhesive used to bond orthodontic brackets to demineralized enamel surfaces. METHODS: Eighty freshly extracted, human maxillary premolars were divided into 4 equal groups and demineralized over the course of 21 days. Brackets were bonded to the demineralized enamel of teeth in Group 1. In Group 2, bonding was performed following resin infiltration (ICON(R), DMG, Hamburg, Germany). Before bonding, pre-treatment with acidulated phosphate fluoride (APF) or solutions containing casein phosphopeptide-amorphous calcium phosphate with 2% neutral sodium fluoride (CPP-ACP/wF) was performed in Groups 3 and 4, respectively. The SBS values of the brackets were measured and recorded following mechanical shearing of the bracket from the tooth surface. The adhesive remnant index (ARI) scores were determined after the brackets failed. Statistical comparisons were performed using one-way ANOVA, Tukey's post-tests, and G-tests. RESULTS: Significant differences were found in some of the intergroup comparisons of the SBS values (F = 39.287, p < 0.001). No significant differences were found between the values for the APF-gel and control groups, whereas significantly higher SBS values were recorded for the resin-infiltrated and CPP-ACP/wF-treated groups. The ARI scores were also significantly different among the 4 groups (p < 0.001). CONCLUSIONS: Tooth surfaces exposed to resin infiltration and CPP-ACP/wF application showed higher debonding forces than the untreated, demineralized surfaces.

Correlation study on bone density, microstructure and acoustic parameters of cancellous bone at different stages of decalcification / 医用生物力学

Objective To study changes and correlations in ultrasound acoustic parameters, bone density and microstructure of the cancellous bone at different stages of decalcification. Methods Fifteen defatted porcine cancellous bone specimens were decalcified at different decalcification stages, and the bone density, microstructure and acoustic parameters were measured by Micro CT and ultrasound system, respectively, before and after the decalcification. Correlations between acoustic parameters, bone density and microstructure were investigated. Results With the loss of calcium in bone specimens, BMD (bone mass density), BS/TV and BV/TV decreased continuously. Microstructure parameters SMI and BS/BV increased, while Tb.Th and Tb.N decreased with Tb. Sp increasing. Degree of anisotropy (DA) increased. Acoustic parameter SOS increased at first, and then decreased, with nBUA slightly decreasing. High correlation was found between acoustic parameters, BMD and bone microstructure parameters. Conclusions Ultrasound acoustic parameters are correlated with BMD and bone microstructure. This study may provide some reference information for the early diagnosis of osteoporosis based on ultrasound.

Correlação entre a avaliação microscópica e a leitura por laser fluorescência de lesões de manchas brancas em dentes bovinos: estudo in vitro / Correlation between the microscopic evaluation and reading by laser fluorescence of enamel decalcification in bovine teeth: in vitro study

Objetivo: avaliar a correlação entre as leituras de descalcificação artificial de esmalte in vitro por meio do DIAGNOdent® e pela microscopia óptica com luz polarizada. Metodologia: foram utilizados 25 dentes bovinos que foram expostos a um meio artificial de cáries por uma hora, duas vezes ao dia, por 35 dias. Em seguida, procedeu-se as leituras das fluorescências utilizando o aparelho laser DIAGNOdent®. Foram registradas as leituras das áreas sem descalcificação (para calibração) e as áreas desmineralizadas artificialmente. Após as leituras da fluorescência, as regiões de leitura por laser foram cortadas em secções de aproximadamente 400μm e avaliadas por meio da microscopia de luz polarizada. Resultados: demonstraram que, embora com algum grau de variação, o sistema DIAGNOdent® foi capaz de detectar as descalcificações semelhantemente àqueles reconhecidos por microscopia de luz polarizada, estabelecendo um padrão razoável de equivalência de leituras. Conclusão: os valores médios encontrados para o DIAGNOdent® demonstraram um coeficiente de correlação de Pearson de 0,63 com relação aos valores das leituras microscópicas.

Aim: evaluate the correlation between the artificial in vitro enamel decalcification through DIAGNOdent® laser and through optic microscopic with polarized light. Methodology: for this research, 25 bovine teeth had been exposed to an artificial decay environment during one hour, two times a day, for 35 days. After wards, DIAGNOdent® laser has been used to read the fluorescence from the enemel areas without decalcification (for calibration) and to evaluate the non-mineralized artificially. After the fluorescence readings regions of laser read were cut into sections of about 400μm and evaluated by polarized light microscopy. Results: the results had demonstrated that, even with some degree of variation, the DIAGNOdent® system was capable of recognizing the decalcification, establishing regular equivalence of standard readings such as light polarized microscopy. Conclusion: the average values found for DIAGNOdent® laser had demonstrated a 0,63 coefficient of correlation of Pearson to the values of microscopically readings.

Preservação da proteína verde fluorescente no tecido ósseo descalcificado / Preservation of the green fluorescent protein on decalcified bone tissue

A proteína verde fluorescente (GFP) foi originalmente descoberta no cnidário Aequorea victoria. Células-tronco GFP positivas podem ser rastreadas in vivo quando usadas na terapia de doenças. No entanto, no osso, a fluorescência gerada pela GFP pode ser perdida durante o processo de descalcificação, dificultando o rastreamento das células-tronco usadas no tratamento de doenças ou defeitos ósseos. O objetivo deste estudo foi comparar diferentes técnicas de preservação da GFP no tecido ósseo descalcificado. Foram utilizados fêmures de ratas GFP Lewis distribuídos em quatro grupos: 1) descalcificado em ácido fórmico e incluído em parafina; 2) descalcificado em ácido fórmico e submetido à criomicrotomia; 3) descalcificado em EDTA e incluído em parafina; e 4) descalcificado em EDTA com criomicrotomia. Secções de tecido ósseo de todos os grupos foram analisadas para identificação da fluorescência natural e posteriormente submetidas à imunofluorescência, sendo utilizados anti-GFP e Alexa Flúor 555. As imagens foram obtidas por microscopia confocal. Osteócitos, osteoblastos e células da medula óssea de ratos GFP somente tiveram sua fluorescência natural preservada no tecido ósseo descalcificado em EDTA e submetido à microtomia por congelação. Nos demais grupos, houve perda da fluorescência natural, e as células GFP somente puderam ser identificadas com o uso da reação de imunofluorescência com anti-GFP. Conclui-se que a descalcificação em EDTA e a criomicrotomia são as melhores técnicas para preservar a fluorescência natural das células GFP no tecido ósseo e que a visualização de células GFP em tecido ósseo descalcificado em ácido fórmico e incluído em parafina somente pode ser realizada com o uso da técnica de imunofluorescência.

Green fluorescent protein (GFP) was originally derived from the cnidarians Aequorea victoria. GFP-positive stem cells can be tracked in vivo when used in the therapy of diseases. However, in the bone, the fluorescence generated by GFP can be lost during the decalcification process, hindering the tracking of stem cells used in the treatment of diseases or bone defects. The aim of this study was to compare different techniques of preservation of GFP in the decalcified bone tissue. Femurs of female Lewis GFP rats were distributed in four groups: 1) decalcified in formic acid and paraffin-embedded; 2) decalcified in formic acid submitted to cryomicrotomy; 3) decalcified in EDTA and paraffin-embedded and 4) decalcified in EDTA with cryomicrotomy. Sections of bone tissue of all the groups were analyzed for identification of the natural fluorescence and subsequently submitted to the immunofluorescence using anti-GFP and Alexa Flúor 555. The images were obtained by confocal microscopy. Osteocytes, osteoblasts and bone marrow cells of GFP rats only had natural fluorescence preserved in the bone tissue decalcified in EDTA and submitted to cryomicrotomy. In others groups there were loss of the natural fluorescence and the GFP cells could be only identified with the use of the immunofluorescence with anti-GFP. In conclusion, the decalcification in EDTA and the cryomicrotomy are the best techniques to preserve the natural fluorescence of the GFP cells in the bone tissue and the GFP cells in bone tissue decalcified in formic acid and paraffin-embedded can be visualized only with the use of the immunofluorescence with anti-GFP.

Estudo comparativo da morfologia da polpa dentária de dentes humanos submetidos à técnica de clivagem e a técnicas de descalcificação química / Comparative study of dental pulp morphology from human teeth submitted to cutting technique or chemistry decalcification technique

Os rápidos avanços do conhecimento acerca do reparo e regeneração tecidual, têm despertado o interesse pela biologia pulpar. Entretanto, para avaliar microscopicamente a dinâmica do tecido pulpar, é necessário, inicialmente, que o dente seja submetido aos processamentos histológicos de fixação e descalcificação. A descalcificação pode afetar o grau de coloração e pode causar desnaturação de proteínas. Além disso, é um processo demorado, visto que o dente requer um longo período de desmineralização. Assim, a proposta deste trabalho foi de avaliar qualitativamente a matriz extracelular e as células da polpa dentária, comparando três grupos: dois em que o tecido dentário foi descalcificado e um em que a polpa foi removida dos tecidos duros, não necessitando do processo de descalcificação. Dez pré-molares foram fixados em formalina tamponada a 10% por 24 horas. Após, estes dentes foram divididos em três grupos: 4 dentes foram submetidos a processo de descalcificação por meio de solução de Morse, 3 por meio de solução de EDTA a 10% e; os 3 dentes restantes tiveram sua polpa separada dos tecidos duros dentários por meio da técnica de clivagem. Na seqüência, os três grupos foram processados por meio da técnica histológica de rotina e foram corados com H/E. Os resultados desta análise demonstraram que houve uma melhor conservação tanto da matriz extracelular, quanto das estruturas celulares no grupo da clivagem, seguido do grupo Morse e por fim, com a menor conservação das estruturas pelo grupo EDTA.

The rapid advances of the knowledge of repair and regeneration tissues had proved to be an exciting time for pulp biology. However, to study the dynamic of pulp tissue, it is necessary, initially, that the tooth be submitted to histological fixation and decalcification processing. Decalcification may affect the degree of staining and it may cause denaturation of proteins. Furthermore, it is a slow process, demanding long demineralization times for a tooth. Thus, the purpose of the present study was to compare, qualitatively, the pulp extracellular matrix and the pulp cells, submitted to different techniques: EDTA solution decalcification, Anna Morse solution decalcification and a last group which pulp was removed from tooth without decalcification. Ten premolar teeth were fixed in 10% buffered formalin for 24 hours. After this, the teeth were divided in three groups: 4 teeth underwent decalcification with Morse solution; 3, decalcification with 10% EDTA solution and; 3, were sectioned and their pulps were gently removed. Subsequently, the groups followed the routine histological technique and staining with H/E. The results demonstrated that both conservation of pulp cells and extracellular matrix were better in the group without decalcification, followed by the Morse group and, the last, with the worst structures conservation for the EDTA group.