Histochemical study of dentine in estrogen-treated rats

Research

Histochemical study of dentine in estrogen-treated rats Sol Be&&,

Ph.D., and Benjamin

H. Ershoff, Ph.D., Los Angeles

ad

Culver City, Calif. DEPARTMENT CALIFORNIA,

A

OF ANATOMY, AND

WESTERN

SCHOOL

OF MEDICINE,

BIOLOGICAL

UNIVERSITY

OF SOUTHERN

LABORATORY

INTRODUCTION

number of studies have shown that the administrat,ion of estrogenic hormones to rats produced an inhibition of linear growth as well as an osteosclerosis-like appearance to the shafts of the long bones.lS3 It was thought by these workers that the accretion of bone in the shaft was due to a failure in the resorption and remodeling of the primary trabeculae. We administered estrogenie hormones to both male and female rats and were able to show by histochemical methods that the ground substance of the primary trabeculae was altered in such a manner as to interfere with normal calcification.4 As the previous studies were mainly limited in scope to the action of estrogen upon the growing apparatus, it was concluded that the effects of the hormone were limited to a specific action upon the endochondral growth of bone. On the other hand, little attention has been directed toward other types of bone formation, such as intramembranous formation. Therefore, the present study was undertaken to add data which may help to explain the influence of estrogen upon other calcifying processes. Since the composition and calcification of dentine are similar in many ways to intramembranous bone formation, dentine was selected for this study. MATERIALS

AND

METHODS

Thirty male and thirty female rats of the Holtzman strain, averaging 44 grams in weight, were selected at weaning, and the animals of each sex were This Institute Service.

investigation was of Dental Research

supported by of the National

Research Institutes

Grant D-1033 (C-2) of Health, United

from States

the National Public Health

249

250

IIernick

and

Ershof

0,s.. O.M. & 0.I’. ~\ugust,

1 wk

divided into three groups. The rats ill (:roup 1 scrvctl IIS nont rcatctl tout rols ; the animals in Groups 1-I and IIL wcrc’ given subcutauc~ousinjections, six times weekly, of 100 pg e&radio1 henzoatc in 0% cc. ses:~mcoil and 100 pg diethylstilbestrol in 0.25 CC. sesameoil, respectively. A natural food stock ration (Rockland rat diet) and water were provided ad libitum. Six rats in each group were found to suffice for a qualita,tivc evaluation. They were autopsied after 37 weeks of treatment. The heads wcrc removed and fixed in an alcohol-acctie acid-formalin solution, after which they were decalcified in a 10 per cent solution of nitric acid in 10 per cent formalin, dehydrated, and infiltrated in the routine manner for nitrocellulosc embedding. The sections wcrc cut. at 20 microns, and alternate sections were sta,ined with hrmatoxylin and triosin, Mallory’s connective tissue stain, alcian blue-periodic acid-Schiff stain, and toluidine blue for metachromasia. RESULTS

The organic matrix of dentine is made up of collagenous fibrils and a ground substance consisting of a protein-mucopolysaccharide complex. In the process of calcification, the organic mat,rix becomes calcified in the form of globules, the calcospherites, which, when fused together, give the dentine a homogenous appearance. Fig. 1 illustrates a hematoxylin and triosin section of the incisal dentine from a nontreated control animal. Two layers are distinguished: (1) a wide outer zone, which is homogenous in appearance and represents the mature calcified dentinc, and (2) the inner region, the uncalcified predentine, which stains light pink. When there is a disturbance in calcification, the calcospherites fail to fuse, producing a “splotchy” appearance that is characteristic of interglobular dentine. The dentine from the estrog’cntreated animals exhibited an increased number of t,hese acidophilic poorly calcified areas (Fig. 2). HISTOCHEMICAL Metachromasia

STUDIES

In the control animals only the predentine layer stained a faint violet after the sections were exposed to toluidine blue, whereas the calcified dentine failed to show metachromasia (Fig. 3). In contrast, the dentine of the estrogen-treated animals stained differently with toluidine blue. There was an increase in metachromasia. Both the predentine and the dentine appeared violet in color, with the interglobular spaces staining more intensely (Fig. 4). Alcian

blue-PAS

reaction

The staining of sections with an alcian blue-periodic acid-Schiff combination permits the differentiation between the acid and neutral mucopolysaccharides. Fig. 5 shows a section of incisal dentine from a nontreated control animal that was exposed to this staining combination. The positive alcian blue material is mainly associated with the tubules, while the remaining portion of the dentine appears pink in color. In contrast, the dentine from the estrogentreated animals exhibited an abundance of positive alcian blue material which

Volume Number

18 2

Dentine

in estrogen-treated

rats

251

Fig. 1. Incisal dentine from a control animal. Note the homogenous appearance of the calcified dentine. (Hematoxylin and triosin stein. Magnification, x250; reduced I/&,.) Fig. 8. Incisal dentine from an estrogen-treated animal. Note the increase in the number of intergIobular areas in the dentine. (Hematoxylin and triosin stain, Magnification, x250; reduced r/lo.) Fig. 3. Tncisal dentine from a control animal. The section was treated with toluidine blue for metachromasia. Note that predentine only exhibits pale metachromatic staining. (Magnification, x.250; reduced rho.) Fig. 4. Incisal dentine from an estrogen-treated animal. The section was treated with toluidine blue for metachromasia. The dentine now exhibits metachromasia with interglobular areas showing a more intense reaction. (Magnification, x250; reduced xo.) Fig. 5. Incisal dentine from a control animal. The section was stained with an alcian blue-PAS combination. Note that the positive aleian blue material is associated with the tubules. The remaining portion of the dentine appeared pink in color. (Magnification, x250; reduced I/&.) Fig. 6. Incisal dentine from an estrogen-treated animal. The section was stained with an alcian blue-PAS combination. Note the increased amount of positive alcian blue material localized mainly in the incremental lines. The spaces between the incremental lines stained deep red in color. (Magnification, x250; reduced &.)

252

Bernick

and

EwhofS

O.k. ox. August,

was mainly localized in the incremental lines. The intertubular the positive alcian blue regions stained deep red (Fig. 6). Alkaline

phosphatase

& 0.1’. 1961

areas bet,wecn

reaction

The odontoblastic layer and the dentine from the control animals responded in an intense manner to Gomori’s cobalt stain (Fig. 7). To distinguish between preformed and enzymatically deposited phosphate, alternate cut sections from the control a,nimals were treated by the same method but the enzymatic substrate was omitted. The negative results obtained under such conditions are seen in Fig. 8. The administration of estrogen appeared to alter the reactions of the odontoblasts and dentine to Gomori’s technique (Fig. 9), The odontoblasts gave a negative response, while dentine stained lessintensely than the control dentine. DISCUSSION

The process of dentinogenesis is initiated by the appearance of an uncalcified matrix composed primarily of collagenous fibrils and a ground substance consisting of a protein mucopolysaccharide complex. The composition and state of the ground substance have been shown to exert an influence upon calcification. Rubin and Howard,5 Neuman and associates,6and Sobe17demonstrated that a relationship exists between the acid mucopolysaccharides and the calcium phosphate binding. The mineralization of the dentine does not occur uniformly throughout but aggregates into isolated rounded clusters of calcium apatite crystals, the calcospherites. The calcospherites eventually tend to coalesce into a homogenous incremental layer of dentine. The failure of these aggregates to fuse results in the production of acidophilic interglobular spaces.Any alteration in the formation of the dentine by dietary or hormonal disturbances will result in an increased number of these poorly calcified interglobular spaces. In the present investigation the dentine of the estrogen-treated animals exhibited an abnormally high number of these spaces. Inasmuch as the mineral intake was unaltered, one may assume that the disturbance in calcification may be related to changes in the organic matrix. It is generally accepted that the mucopolysaccharides exhibit metachromasia with basic aniline dyes, such as toluidine blue, and give a positive reaction with both alcian blue and periodic acid-Schiff stains. These staining reactions not only demonstrate the presence of the mucopolysaccharides in bone but also reveal the degree of polymerization.8 Levene and Schubert9 demonstrated by chemical methods that an increase in calcium ions in combination with chondroitin sulfate reduces or abolishes metachromasia. SiffertlO found that the metachromatic staining of calcified cartilage disappears as calcification takes place. When the periodic acid-Schiff method is applied to fully calcified dentine or bone, the ground substance is barely stainable, indicating that it contains only highly polymerized mucopolysaccharides.ll-ls These same investigators also showed that immature or poorly calcified dentine or bone stained more intensely ; to them, this was indicative of a lesser degree of polymerization. In the present study the histochemical results from the control

Volume Number

18 2

Dentine

in estrogen-treated

rats

253

Fig. 7. Control section of incisal dentine that was stained by Gomori’s alkaline phosphatase method. Note intense positive reaction in dentine and odontoblastic layer. (Magnification, x250; reduced go.) Fig. 8. Control section of incisal dentine that was incubated without glycerophosphate. Compare with Fig. 7. Note the absence of response in dentine and pulp. (Magnification, x250 ; reduced yro.) Fig. 9. Section of ineisal dentine from estrogen-treated animal that was stained by Gomori’s alkaline phosphatase method. Note the decrease in staining intensity of the dentine and the negative response of the odontoblasts. (Magnification, x250; reduced ?T”~.)

animals agree with the findings 01’ t hca prc~viousl~. mcntion(ld ~~orkt~t+s, 1hat is. the dcntinr stained pink with ~(~z’iotlic~ aeicl-HchiW stain illld show-otl only isolated positive a,lcian blue areas which also stained faintly violet with toluidine blue. On the other hand, the estrogen-treated dentine appeared deep red in color and exhibited an abundance of positive alcian blue regions which were metachromatic after toluidine blue staining. These staining responses would imply that the ground substance cont,ains mucopolysaccharides in lesser dcgrce of polymerization, a condition predisposing to an interference in calcification. Priest and co1leagues14 administerrd estradiol to rats and Pound that this hormone inhibited the synthrsis of acid mucopolysaccharidcs. They based their conclusions on the fact that there was a reduction in thrl incorporation of SHG into cartilage and the aortas. Applying t’hc findings of the prcsctnt investigation and the observations reported by other workers, one may coucelude that, estrogen, by its action upon the acid mucopolysaccharidcs of the ground substance of dentine, alters its receptive state, thus inhibiting proper calcification. The role of alkaline phosphatase in the process of calcification is one which to this day is not clearly dcfincd. However, the enzyme has been localized in both the odontoblastic la-er and dentine.‘“, “j In the present study differences were noted between the dentine of the control animals and that of the other experimental animals in thrir reaction to Gomori’s technique. The extent to which these differences are related to the presence of alkaline phosphatase is questionable, inasmuch as the specimens were fixed in an alcoholacetic acid-formalin solution and decalcified in nitric acid, procedures which should inactivate the enzyme. On the other hand, the dentine from the control sections that were incubated without, glyccrophosphate and from the estrogentreated animals gave a ncgativc or a faint response, respectively, whereas the control sections stained in a positive manner following treatment with QOmori’s method. SUMMARY In the present investigation, histologic sections of t,he teeth from cont,rol and estrogen-treated rats were stained by various histochemical methods in an effort to determine the influence of estrogenic hormones upon the calcification of dentine. The estrogen-treated dentine stained deep red with periodic acid-Schiff stain and exhibited an abundance of positive alcian blue material which was also metachromatic in nature. These staining reactions would indicate that there was an alteration in the ground substance. This situation produced an interference in the calcification of the dentine. REFERENCES

1.

Silberberg, M., and Silberberg, R.: Steroid Hormones and Bone. In Bourne, G. H. (editor) : The Biochemistry and Physiology of Bone, New York, 1956, Academic Press, Inc., chap. XX, pp. 623-668. 2. Budy, A. M., Uriut, M. R., and McLean, F. C.: The Effect of Estrogens on the Growth Apparatus of the Bone of Immature Rats, Am. J. Path. 28: 1143, 1952. 3. Day, H. G., and Follis, R. H., Jr.: Skeletal Changes in Rats Receiving Estradiol Benzoate as Indicated by Histological Studies and Determinations of Bone Aah, Serum Calcidm and Phosphatase, Endocrinology 28: 83, 1941.

Volume Number

18 2

Dentine

ilb estrogen-treated

rats

255

4. Bernick, S., and Ershoff, B. H.: Histoehemical Study of Bone in Estrogen-Treated Rats, J. D. Res. 42: 981, 1963. 5. Rubin, P. S., and Howard! J. E.: Histochemical Studies on the Role of Acid Mucopolysaccharides in Calcifiability and Calcification, Tr. Macy Conf. on Metabolic Interrelations 2: 155, 1950. 6. Neuman, Properties of Cartilage, W. F., Boyd, E. S., and Feldman, I.: The Ion-Binding Tr. Macy Conf. on Metabohc Interrelations 4: 100-112, 1952. 7. Sobel, A. E.: Studies on the “Local Factor” of Calcification, Tr. Macy Conf. on Metabolic Interrelations 4: 113, 1952. 8. McLean, F. C., and Urist, M. R.: Bone, ed. 2, Chicago, 1961, University of Chicago Press, pp. 35-44. 9. Levene, A., and Schubert, M.: Metachromasia of Thiazine Dyes Produced by Chondroitin Sulfate, J. Am. Chem. Sot. 74: 91, 1952. 10. Siffert, R. 8.: The Role of Alkaline Phosphatase in Osteogenesis, J. Exper. Med. 93: 415, 1951. 11. Bevelander, G., and Johnson, P. L.: A Histochemical Study of the Development of Bone. Anat. Rec. 108: 1. 1950. 12. Engel, M.: Glycogen and Carbohydrate Protein Complex in Developing Teeth of the Rat, J. D., Res. 27: 681, 1948. 13. Heller-Steinberg, M.: Ground Substance, Bone Salts, and Cellular Activity in Bone Formation and Destruction, Am. J. Anat. 89: 347, 1951. 14. Priest, R. E., Koplitz, R. M., and Bendett, E. P.: Estradiol Reduces Incorporation of Radioactive Sulfate Into Cartilage and Aortas of Rats, J. Exper. Med. 112: 225, 1960. 15. Bevelander, G., and Johnson, P. L.: A Histochemical Localization of Alkaline Phosphatase in the Developing Tooth, J. Cell. & Comp. Physiol. 26: 25, 1949. 16. Harris, E.: Sites of Alkaline Phosphatase in Dentinogenesis, Anat. Rec. 107: 105, 1950.