ACBTE3田中

ACBTE Vol. 3:73-80, 1993

Studies on Time-Marking of Demineralized Paraffin-Embedded Sections of Hard Tissues Using Ethylenediamine-N,N'-di(methylenephosphonic acid) (EDDPO)

Yoshio TANAKA

Department of Dental Pharmacology,
School of Dentistry at Niigata, The Nippon Dental University,
8-1 Hamaura-cho, Niigata 951, JAPAN.

Introduction
Ethylenediamine-N,N'-di(methylenephosphonic acid) (EDDPO) (Fig.1) is a methylenephosphonic acid derivative of ethylenediamine that forms stable chelates with a number of bivalent cations1). We found previously that when EDDPO was injected sequentially at periodic intervals into rats, hematoxylin-stained lines corresponding to the time of each EDDPO injection were produced on paraffin sections of demineralized incisor dentin. This histological observation indicated that EDDPO might be applicable as a time-marking agent for vital staining of hard tissues. The present study was therefore undertaken to establish appropriate experimental conditions for the use of EDDPO as a time marker and to examine the toxicity of EDDPO.
The study consisted of two experiments. The first experiment was done to establish the optimum dosage rate for time marking and the most desirable conditions for histological preparation. The second experiment was done to examine the toxicity of EDDPO, with a view to its practical use.

Materials and Methods
First Experiment
Young male Sprague-Dawley (S.D.) rats (Japan SLC, Inc., Shizuoka, Japan) with an average body weight of 110 g were used in all experiments. The rats were given commercial food pellets (CLEA Japan, Inc., Tokyo, Japan) and tap-water ad libitum. EDDPO (Dojindo Labs., Kumamoto, Japan) was administered to rats by injection subcutaneously or intraperitoneally every other day, in the following doses and order: 32, 8, 14, 20, 26 and 32 mg/kg/day. The EDDPO dissolved in distilled water was adjusted to pH 7.4 with 2N sodium hydroxide and given in a volume of 0.1 ml per 100 g animal weight. Twenty-four hours after the last injection of EDDPO, experimental rats under general anesthesia were perfused and fixed through the right ventricle with three kinds of fixative solutions: 0.1 M phosphate-buffered 3.7% formaldehyde, 4% paraformaldehyde - 2.5% glutaraldehyde, or 2.5% glutaraldehyde. The mandibles were removed and immersed in the same fixative solution as that used for perfusion.
After fixation for 3 days, 1, 2, 3, 5 or 8 weeks, the mandibles were rinsed and decalcified in three kinds of solution: Plank and Rychlo solution, 5% formic acid or 5% neutral EDTA. Specimens of decalcified mandible were dehydrated in increasing concentrations of ethyl alcohol, and embedded in paraffin by the standard method. Transverse sections 6 μm thick were cut on a Jung-type microtome and stained with six types of hematoxylin solution, i.e. Carrazi, Delafield, Gill #2, Harris, Mayer and Weigert's iron hematoxylin (Sigma, St. Louis, U.S.A.).

Second Experiment
Forty male S.D. rats were assigned to the treatment group and injected subcutaneously with EDDPO at a daily dose of 20, 40, 80 or 400 mg/kg for 10 days, and the remaining 10 rats were used as controls. Ten days after EDDPO treatment, both the treatment and control rats were sacrificed and blood samples were taken from the carotid artery. The blood samples were centrifuged and serum was collected. Serum calcium (Ca), inorganic phosphorus (IP) and alkaline phosphatase (AlPase) levels were measured by spectrophotometry (UV-200, Shimazu, Kyoto, Japan).
The right femurs were removed and freed from adhering tissue for determination of wet weight, then dried in a vacuum desiccator for measurement of dry weight. The dried bones were ashed in a muffle furnace for 20 h at 600 ℃ and for 8 h at 650 ℃ to measure the ashed weight, then dissolved in 25% HCl solution. The calcium content of the bone was determined by atomic absorption (AA-670, Shimazu, Kyoto, Japan). The data were expressed as mean ± standard error of the mean. The significance of differences between the experimental group and the control group was determined by Student's t-test.
For histological observation, the mandibles were removed and immersed in 0.1 M phosphate-buffered 3.7% formaldehyde fixing solution. After fixation for 5 weeks, the specimens were washed with running tap-water. The distal half of the right mandible was decalcified in Plank and Rychlo solution, dehydrated in ethyl alcohol, and embedded in paraffin. Transverse sections (6 μm thick) were cut and stained with Carrazi's hematoxylin solution. The distal half of the left mandible was dehydrated and embedded in methyl methacrylate resin (Wako Pure Chemical, Kyoto, Japan). Transverse sections of mandibular incisors were cut to a thickness of 100 μm using a saw microtome (S拡e microtome 1600, Leica, Heerbrugg, Switzerland), ground to a thickness of 60 μm, and subjected to contact microradiography (C-SM, Softex, Tokyo, Japan) (CMR).

Results and Discussion
First Experiment
Decalcified paraffin sections of incisor dentin fixed for 3 days and one week revealed no hematoxylin-stained lines, irrespective of the kind of fixative agent, decalcification solution or hematoxylin solution used. Hematoxylin-stained lines were visualized on specimens fixed for more than 3 weeks, and prolongation of the fixation time enhanced the color development. The clearest and most intense hematoxylin lines were obtained in specimens fixed for 5 and 8 weeks. Similar intensity was described by Yoshiki et al.2), who reported that in order to detect tetracycline (Tc) lines on the decalcified dentin matrix of Tc-administered rats, fixation for more than 5 weeks in neutral formaldehyde solution was necessary. His observation agreed with ours, and suggest that a fixation time of 5 weeks is necessary to avoid the release into the decalcification solution of elements that participate in color development.
Upon comparison of the sections stained with six kinds of hematoxylin solution, clearly defined lines were obtained on sections stained with Weigert's hematoxylin. In addition, this staining solution allowed detection of lines produced by a low dose (8 mg/kg) of EDDPO. Carrazi's solution allowed detection of lines corresponding to doses of more than 14 mg/kg. The other hematoxylin solutions did not detect the stained lines distinctly.

Second Experiment
There were no detectable differences of net weight gain in groups treated with 20 and 40 mg/kg/day EDDPO in comparison with the control group. In the group administered 80 mg/kg/day EDDPO, moderate reduction of body weight gain was observed, and in the 400 mg/kg/day EDDPO group, the reduction was severe. The serum Ca concentration was elevated dose-dependently by EDDPO treatment. Conversely, there was an inverse relationship between the serum IP level and the administered dose (Table 1).
In the bone components, there were marked changes in high-dose EDDPO groups. Wet, dry and ash weights were significantly decreased after treatment with 400 mg/kg/day EDDPO. Small changes in bone components were observed in the 20, 40 and 80 mg/kg/day EDDPO groups (Fig.4). CMR impressions of the dentin of EDDPO-treated rats showed hypomineralized lines at a dose of 40 mg/kg/day EDDPO. At a dose of 80 mg/kg/day, inhibition of mineralization was more evident, especially in the latter half of the experimental period (Fig.5). The inhibition of mineralization in the dentin and femur seemed to be caused by the methylenephosphonate group of the EDDPO molecule as well as 1-hydroxyethylidene-1,1-bisphosphonic acid (HEBP), which combines irreversibly with hydroxyapatite crystals and reduces crystal growth3,4). The elevation of the serum Ca level in the high-dose EDDPO group seems to be caused by the inhibitory action on mineralization5). The two present experiments showed that EDDPO did not exert a toxic effect or inhibitory action on mineralization at the dosage (16-32 mg/kg) required in practice to mark calcifying tissues. Only a few methods for time-marking of paraffin sections have been reported6-10). Therefore we hope that the present method will be of practical use for research on hard tissues.

References

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Explanation of Figures

Fig. 2 Comparison of H-E stained findings at different formaldehyde fixating period from 3 days, 2 weeks and 5 weeks. After fixation, transverse sections of mandibula dentin were demineralized by Plank & Rychlo solution and stained by the Carrazi's hematoxylin solution. The number with arrow on figure shows dosage of administered EDDPO mg/kg. Bar: 50 μm. Hematoxylin-stained lines were visualized on specimens fixed for more than 3 weeks, and prolongation of the fixation time enhanced the color development. The clearest and most intense hematoxylin lines are obtained in specimens fixed for 5 weeks and dosage of 14 mg/kg over.

Fig. 3 Body weight of control and EDDPO administered rats. (●) group of control; (○) group of 20 mg/kg/day of EDDPO; (△) group of 40 mg/kg/day of EDDPO; (▽) group of 80 mg/kg/day of EDDPO; (□) group of 400 mg/kg/day of EDDPO. Each value represents the mean±S.D. Statistical evaluation of difference between EDDPO-treated group and control group was done using Student's t-test. The asterisks "* and ***" indicate P<0.05 and P<0.001, respectively.

Fig. 4 Bone components in rats treated with EDDPO. Statistical evaluation of differences between EDDPO-treated group and control group were done using Student's t-test. The asterisks "* and ***" indicate P<0.05 and P<0.001, respectively.

Fig. 5 Contact microradiograms of the transverse section of undemineralized mandibular incisor dentin of control and EDDPO treated rats. Bar: 100 μm. At the dose of 40 and 80 mg/kg/day of EDDPO, hypomineralized lines were observed. At the dose of 400 mg/kg/day of EDDPO, hypomineralized area is formed from the first EDDPO administration.