*Dimitrius Leonardo Pitol; **Joáo Paulo Mardegan Issa; ***Flávio Henrique Caetano & ****Laurelúcia Orive Lunardi
*Graduate student, Biosciences
Institute- Molecular and Cellular Biology (UNESP), Rio Claro, Sao
** Graduate student, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.
*** Professor of Cellular Biology, Biosciences Institute- Molecular and Cellular Biology (UNESP), Rio Claro, Sao Paulo, Brazil.
**** Professor of Histology, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.
An open access article from Int J. MorphoL, 25(2):309-313, 2007.
SUMMARY: The aim of this article is to present the decalcification process dynamic of mineralized tissue in dogs, teeth and jaw, comparing the traditional decalcification method, immersion, and microwave, immersion followed by irradiation using a domestic microwave oven, accompanying the liberation of calcium through spectrophotometer of atomic absorption. It was used as decalcified agent, EDTA solution or nitric acid. The results showed that with the use of nitric acid (5%), after 15 days, the irradiated fragments could be processed for histological analysis, otherwise the tooth not irradiated need to be submerged for 65 days. The EDTA decalcified action was slower than the nitric acid. The histological observations of the irradiated samples showed an excellent preservation of the morphological characteristics, independently of the decalcified agent used.
KEYWORDS: Dog; Mineralized tissue; Decalcification; Microwaves; Solution.
Considering histological preparations of the mineralized biological samples, the decalcification process has an important role, because the preservation of tissue structures and its relationship are dependent of the quality and speed of the demineralization process occur (Baird et al, 1967; Hornbeck et al., 1986; Clayden, 1952; Efeoglu et al, 2006). Lillie et al, in 1951, considered that the ideal method for the mineralized tissues study being that remove the more quantity of calcium salts. With this objective, most methods and decalcified agents are described in the literature: simultaneous decalcification with posterior fixation by Bouin solution, an example, or using acids (nitric, formic, sulphurous) and its combination, as well as in different conditions of temperature and concentration (Engelbreth-Holm & Plum, 1951; Clayden; Goncalves& Oliverio, 1965). Substances capable to attach with specific metals (chelation agents) as ethylenediamine tetra acetate acid (EDTA) have also been used as decalcified agents. For teeth decalcification, Warshawsky & Moore, 1967 used EDTA isotonic dissolved in phosphate solution, neutral pH. This solution was calculated for the pH maintenance for a few weeks, promoting not only a good decalcification but also performing a better morphologic preservation of the samples.
The microwave irradiation in domestic oven has been used in diagnosis laboratories, when it was performed the tissue processing with notable time reduction. It is currently used for helping the tissue fixation, accelerating this process and preserving the morphologic and antigenic tissue characteristics (Cunningham et al., 2001; Lunardi & Britto-Garcia, 1996; Login et al, 1997; Roncarolieia et al., 1991; Ng & Nge et al., 1992).
Aiming to establish a fast and efficient teeth and other mineralized tissue decalcification, this research shows the results when it was compared two decalcified agents, nitric acid 5% and Washawsky solution (Washawsky & Moore) to 8,5% (EDTA pH 7.4) in two decalcified methods (soaked with shake up process and soaked followed by irradiation in microwave oven) in mandibular teeth dogs.
It was used healthy teeth, provided from Central Animals House of the University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil and according to the rules proposed by the Ethics Committee. The left molars attached to the mandible arch were removed and fixed in formaldehyde 10%, during 24 hours.
Microwave oven standardization. The standardization method used in this research was proposed by Login & Dvorak (1990), in that was used a domestic microwave oven (Panasonic /700W/2450mHZ) that had its rotative plate turned off. A Becker of glass containing lOOmL of distilled water was inserted on the left side ofthe pre-heated oven in maximal potency by 2 minutes. An Agar/Giemsa block (0.5cm), prepared with 5% of agar/agar in saline solution in that was added Giemsa stained solution (Sigma Chemical St Louis, MO), was soaked in 5mL of fixative solution or decalcification solution, in Petri plastic plate, 35mm, inserted in various positions inside ofthe oven and irradiated with 100% potency in different periods of time. The color alteration ofthe Agar/ Giemsa blocks was used to watch the better irradiation focus and the temperature change. Establishing the area with most microwave incidence into the oven, as well as the exposition time, it was performed the decalcification process.
Decalcification. Considering the individual differences on teeth mineralization, it was decided to use the same teeth in the analysis by two decalcification methods. Thus, the teeth were longitudinal sectioned in bucolingual orientation, separated into three fragments and decalcified with nitric acid or Washawsky solution (EDTA 8.5% pH 7.4). The full teeth were initially weighted and sectioned into three samples, and the measures were respectively, 1.7x1.0cm of width and 2.6cm of length. All the fragments showed the same structures, enamel, dentin, alveolar bone and soft tissues. After fixative period, it was inserted 30mL of decalcified solution and irradiated for 1 minute in ice bath microwave, in sequence the decalcified solution was despise and replaced by another for new irradiation period. Each five irradiation periods, one sample ofthe decalcified solution, was collected for calcium dosage in atomic spectrophotometer. It was performed 10 irradiations per day and the sample stayed into the decalcified solution at freezer until the next day. After fixative period, the group A (control) was placed in 30mL ofthe decalcified solution and this was replaced by another after the sample remotion for calcium dosage, each 5 days.
Calcium dosage. The calcium concentration ofthe selected samples was determined by atomic absorvance spectrophotometry (Shimadzu AA680G spectrophotometer).
Microscopic study. After decalcification period, the samples were dehydrated, included in paraffin and the histologic sections stained by Masson Trichrome. In sequence, they were analyzed and photographed usinga photomicroscope Jenaval, Carl-Zeiss, Ober Kochem, Germany.
The samples exposed to microwave promoted an acceleration of the decalcification process using nitric acid and EDTA as decalcified agent (Tables I and II). Using the traditional method, it was necessary 65 days in nitric acid solution, meanwhile the irradiated tooth needs only 15 days (Tables I and II), both of them with the same softly conditions that is necessary for the histological processing. The Warshwsky solution presented a slowly action being that necessary 150 days soaked in solution and 35 days when irradiated.
The morphological preservation using these two decalcified agents was similar, in exception of a little staining modification by EDTA, where the Trichrome Masson had its acidophihc staining increased. Thus, in the figures A and D, EDTA and nitric acid, respectively, showed the dentin/pulp relationship with preservation of the dentinal tubules, odontoblast and pulp connective tissue. In the figures B and C, nitric acid and EDTA, respectively, show the preservation of the mineralized tissue (cement and alveolar bone) and soft tissues (connective and periodontal tissue) at the root region. In the same region, it was photographed with polarized light (Fig. E) showing in the decalcified sample by EDTA the mainly fibers of the periodontium, placed between the cement (above) and fascicular bone (under). The polarized light permitted a better visualization of the dentin tissue, acellular cement, alveolar bone and periodontium fibers (Fig. F).
The decalcified methods, independently of the demineralized agent used, have in common the fact that are accelerated when the solution is shaken, mechanically or electrolitically. Goncalves & Oliverio used an electric decalcification technique, with alternant chain, increasing the decalcification velocity, according to these authors, this process promotes a molecule shaking, resulting an increasing of the decalcification process. The results of this work, using a molecular shaking induced by microwaves, it was observed a similar effect.
Clearly, in 1978, explained that the acceleration process promoted by microwaves is due to the energetic portion of the electromagnetic spectrum that interacts with dipolar molecules provoking a fast oscillation, in this way, increasing the intra and intermolecular movements of the water and of the polar portion of the protein chain, increasing the temperature and resulting in its coagulation. It was observed that, independently of the demineralized agent used, the microwaves accelerated the process and the morphology was preserved. Some authors suggested that the microwaves can induce an elevation of the temperature, increasing the decalcification process by decalcified agent diffusion (Boon & Kok, 1998; Balatona & Loget, 1989;Vongsavane?a et al., 1990; Tornero et al. 1991). As the same time, an increase of the temperature is interesting, but a higher temperature elevation (55 °C and 60 °C) is a disaster for the morphologic characteristics preservation (Balatona & Loget; Boon & Kok; Tornero et al.).
The calcium lost occurred fastly followed by swell and hydrolysis of the calcified matrix (Lillie et al; Wagenaar et al., 1993). Low et al., 1994, observed that the decalcification process at 55 °C will result in cellular destruction. Aiming to solve this problem it was used ice baths, slowing the temperature, which is maintained around to 38 °C. The spatial distribution of the electromagnetic field and the microwave energy in the oven are not uniform, resulting in hot and cold regions (Login & Dvorak, 1990,1994). Some indirect methods have been used to evaluate the electromagnetic field (Boon & Kok; Login & Dvorak, 1990). It was used, in this study, Agar + Giemsa pigment as it was proposed by Login & Dvorak (1994), a simple and efficient method that permits to detect the region with high intensity of electromagnetic field.
The movement of the samples can affect the standardization and calibration of the microwave oven, thus the rotative plate of this microwave oven was turned off. The results of this research are comparable with other studies that evaluated the microwave use in decalcification process (Balatona & Loget; Vongsavan et al. ; Rode et al., 1996). In all of these cited works, the microwave accelerated the tissue decalcification, but in none of them, it was quantified the discharged calcium.
In this research, the analyses of calcium dose showed that, the period of time between the first 6 hours until the second day of irradiation occurred the highest concentration of discharged calcium.This discharged calcium increased during the first six hours, which corresponds to enamel decalcification, otherwise, the discharged calcium at the second day, corresponds to the mandibular bone decalcification, which was slower than the first process. The sample maintained only in solution immersion presented an increasing of the discharged calcium corresponded to the enamel decalcification, only at thirty five day.
Under light microscopy study it was not possible to detect morphological alterations produced during the decalcification process, when it was compared a strong acid, like nitric acid to chelation agent, like EDTA. It is possible that exists an ultrastructurally differences between these two decalcified agents, independently of the decalcification speed.
Thus, it is possible to conclude that if we follow some parameters, like good tissue fixation, microwave oven standardization and temperature decrease, with ice baths, we will achieve a morphologic preservation of the mineralized tissues, independently of the decalcified agent used.
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Figure A Photomicrograph of the stained tooth (Masson Trichrome) and decalcified by EDTA showing the good preservation of the dentinal tubules (1), inflammatory cells (2), pulp tissue (3).Photomicrograph of the stained tooth (Masson Trichrome) and decalcified by EDTA showing the good preservation of the dentinal tubules (1), inflammatory cells (2), pulp tissue (3).
Figure B Photomicrograph of the stained tooth (Masson Trichrome) and decalcified by nitric acid 5% showing the good preservation of the dentinal tubules (1), inflammatory cells (2), pulp tissue (3).
Figure C Representative photomicrograph of the stained mandible (Masson Trichrome) and decalcified by EDTA showing the good preservation of the cement (1), periodontal ligament of the root (2), alveolar bone (3).
Figure D Photomicrograph of the stained mandible (Masson Trichrome) and decalcified by nitric acid 5% showing the good preservation of the cement (1), periodontal ligament of the root (2), alveolar bone (3).
Figure E Photomicrograph of the mandible with the tooth stained by Masson Trichrome showing the good preservation of the dentin tissue (1), acellular cement (2), periodontal ligament of the root (3) and bone tissue (4).
Figure F Representative photomicrograph of the mandible with the tooth stained by Masson Trichrome showing the good preservation of the dentin tissue (1), acellular cement (2), alveolar bone (3) and periodontium fibers (4).