Arch. oral Bid. Vol. I. pp.97-105, 1959. Pergamon Press Ltd. Printed in Gt. Britain.

PIGMENTATION OF THE ENAMEL OF ALBINO RAT INCISOR TEETH*+

G. STEIN and P. E. BOYLE

School of Dental and Oral Surgery, Columbia University, and School of Dentistry, Western Reserve University, Cleveland, Ohio U.S.A.

Abstract-The yellow pigment of the incisor tooth of the normal albino rat becomes visible at approximately the midpoint of the convex, enamel-covered surface. Pigmenta- tion occurs in an area incisal to the region where full thickness of the enamel has been attained and after complete acid solubility (maturation) has occurred. The pigment is confined to the outermost zone of the enamel. This zone is relatively acid resistant as compared to the underlying unpigmented portion of enamel.

Prior to the appearance of pigment in the enamel, the overlying enamel organ contains yellow pigment granules. Concurrently with the deposition of pigment into the enamel the granules disappear from the ameloblasts.

When the pigment-containing part of the enamel organ is surgically destroyed no pigment is deposited in the underlying enamel but the enamel surface is not otherwise affected.

A primary cuticle is described as present over the entire surface of the enamel except for the proximal zone where enamel matrix is being deposited. The possible role of this cuticle in calcification (maturation) and sideration (pigmentation) is discussed.

ALMOST half a century ago ERDHEIM (1911) observed that experimental hypopara- thyroidism caused loss of pigment and the appearance of an opaque enamel surface in rat incisors. Since that time there have been published numerous reports on enamel- pigmentation disturbances due to nutritional deficiencies, hormonal disturbances, and intoxications by various substances (see PINDBORG, 1947, and TORELL, 1955, for reviews of the literature).

STEIN and BOYLE (1941) stated that the yellow or deep-orange pigment present in the outer part of the enamel appears approximately midway (Fig. 2) from the formative to the incisal end of the rat incisor, i.e. in the region where the full width of the enamel has been formed and where calcification has progressed to a stage where all or almost all the enamel disappears upon decalcification (Figs. 3 and 4). Upon histologic observation yellow pigment in the form of granules is observed in ameloblasts and the cells of the stratum intermedium beginning at about the border between the proximal and middle thirds of the tooth (Figs. 8 and 10). The pigmented enamel layer becomes relatively resistant to the action of 5% nitric acid (Figs. 4

* All experimental procedures were performed at the Harvard School of Dental Medicine prior to 1944.

t This project was supported in part by a grant D-747((X) from the National Institutes of Health.

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98 G. STEIN AND P. E. BOYLE

and 5). Through decalcification of the stained part of the incisor, two.membrane-like structures are demonstrable: (1) an outer, primary cuticle approximately 1 p in thickness and (2) a yellow layer of enamel approximately 8 p in thickness. Moreover, surgical destruction of the enamel organ in the proximal third of the tooth resulted in spots of uncoloured enamel observed upon subsequent eruption of the affected tooth structure (Fig. 14). Certain of our findings have been confirmed by others (BUTCHER, 1953; WEINMANN, 1943; PINDBORG, 1947, 1953).

The present report presents a detailed description of the material upon which our original abstract was based (STEIN and BOYLE, 1941).

MATERIAL AND METHODS

Incisors of thirty-eight, chiefly adult, rats have been examined. Most observations were made either on undecalcified cross and longitudinal sections cut with a ‘lightning’ disc rotating under water at high speed and subsequently ground by hand. In order to preserve the enamel organ in situ the specimens were fixed in 10% formalin. They were not allowed to become dry in any stage of preparation. For the same reason grinding by hand was performed at a slow rate.. The ground sections were dehydrated, embedded in balsam and for the most part left unstained. Certain sections were decalcified, using either 5 % or 2 % aqueous solution of nitric acid or 5 % sulpho- salycylic acid. and the process of decalcification was observed microscopically. Corresponding parts of the tooth of the opposite jaw were decalcified, embedded, cut in serial sections (transverse and longitudinal) and mounted for microscopic examination. In other animals the enamel organ was removed from the underlying enamel, embedded and sectioned without decalcification. In still another group, the soft enamel matrix at the formative end of the tooth was removed together with the overlying enamel organ and sectioned without decalcification. Unstained sections as well as sections stained by haematoxylin and eosin, phosphotungstic acid and haematoxylin, Mallory’s aniline blue connective tissue stain, Turnbull’s and Berlin blue were examined. In a few instances the yellow outer layer of the enamel was isolated during short-time decalcification by means of 2% nitric acid under the dissecting microscope and then embedded and sectioned. Undecalcified sections of teeth were X-rayed and incinerated in an S. S. White electric oven designed for fusing porcelain.

In addition the enamel organs of twelve teeth were surgically damaged in various parts of the incisor in an endeavour to learn more about the function of the ameloblasts.

OBSERVATIONS

The ground sections prepared were approximately 100 p in thickness. The enamel organ and the periodontal tissue between the tooth and the surrounding alveolar bone were usually preserved in approximately normal relations in the cross sections. When attempts were made to prepare longitudinal ground sections of the incisor tooth in situ, the enamel organ and overlying alveolar bone often separated from the tooth surface as the section thickness approached the goal of 100 CL. The strength of attachment of the enamel organ to the enamel surface was found to vary in different

PIGMENTATION OF THE ENAMEL OF ALBINO RAT INCISOR TEETH 99

regions of the tooth. This is in accord with the observation of others (KALNINS, 1952) that in the constantly growing teeth of rodents, the junction between enamel organ and enamel matrix is unstable and vulnerable not only post mortem, but also intra vitam. It was difficult to maintain the connexion between the ameloblasts and the enamel surface during the removal of the surrounding bone in the region where enamel matrix was being deposited. The adherence between the ameloblasts and the enamel surface increases where the yellow pigment is present in the ameloblasts, and it is greatest before the yellow stain occurs in the enamel proper (Fig. 9).

According to BOYLE and KALNINS (unpublished) the curvature of the rat upper incisor can be compared with the dial of a watch between 9 and 4 o’clock. From Fig. 1 we can see that the growth and maturation of enamel matrix occurs in the portion corresponding to 9-10.45; the tooth erupts from the alveolus (labial alveolar crest) at a point corresponding to 1.30; and it appearsin the mouth cavity before 2.30.

In a longitudinal ground section of the upper incisor of an adult rat the yellow substance in the ameloblasts (Figs. 8, 9, and 10) may first be observed in the area corresponding to 10.45-l 1 (at approximately 7-9 mm from the formative end). Since the rate of growth of the upper incisor is approximately 2 mm a week, this corresponds to the fourth week after the beginning of enamel matrix formation of the particular ameloblast .

The area where the yellow pigment disappears from the ameloblasts and appears in the outermost layer of the enamel varies. In teeth measuring 25-27 mm in total length, this transition area was found to be lo-15 mm from the formative end. This takes place at the fifth to eighth week following initiation of enamel matrix formation of the particular cell, corresponding to 12-12.45. Within a half week (1 mm) this yellow substance disappears from the ameloblasts and appears in the fibrous part of enamel (Fig. 9).

The yellow stain shows a strongly positive reaction to Turnbull’s blue and Berlin blue (Fig. 11). It tends to disappear from the cells during decalcification. The pigmented enamel layer becomes brick red upon incineration. The pigmented layer is relatively acid resistant. This property is shown when nitric acid is applied to ground sections. Whereas the non-pigmented portion of enamel becomes dissolved in a few minutes, the pigmented layer becomes dissolved within 3-4 hr (Figs. 4 and 5). After application of acid the whole yellow layer peels off. It is rather brittle and it is not easy to get a continuous sheet of it.

That formation of the pigmented layer occurs due to alteration of the outer zone of enamel but not due to the addition of a new layer (as in the case of cuticle) is established by measurements of cross sections. There is no measurable increase in thickness of the enamel due to the appearance of the pigmented layer. The pigmented layer is 6-8 II_ in thickness and shows a pattern similar to the arrangement of the ends of enamel rods (Fig. 6). This pattern is caused by short projections from the inside of the layer. These projections become visible on the inner surface of the pigmented enamel when decalcification of the acid soluble portion of enamel is complete.

There is a colourless and structureless membrane, the enamel cuticle, between the enamel and the ameloblasts (Fig. 12). This cuticle appears as soon as the full

loo G. STEIN AND P. E. BOYLE

thickness of enamel matrix becomes established and it persists unchanged to the incisal edge. No change in the appearance of the cuticle could be noted following the passage of the yellow substance from ameloblasts into enamel. By the action of acid alone the pigmented layer of enamel and the cuticle do not always separate from each other distinctly. Sometimes this separation could be obtained by tapping slightly with the point of a dissecting needle against the coverglass during the process of decalcification. It was noted that the adherence of the cuticle to the enamel surface is stronger than to the ameloblasts. If the ameloblasts have become detached from the enamel during preparation of the specimen, the membrane remains in connexion with the enamel surface. However, it can then be separated from the enamel surface by decalcification. This separation takes place only in those regions where the enamel is acid soluble. Near the formative end a region is reached where the enamel is not as yet of full thickness and does not disappear under the action of acid. No cuticle is demonstrable in this region.

If the enamel organ is surgically destroyed in an area where the cuticle is present and where the pigment is still concentrated in the enamel organ (yet not deposited into enamel), a non-pigmented portion of the tooth eventually erupts into the mouth cavity. This is soon followed by normal yellow stained enamel (Fig. 14). The enamel surface contour is not affected.

DISCUSSION

It has been stated by other’workers that when the full width of the enamel is complete, the last product of ameloblasts is the primary cuticle (NUCKOLLS, LEICESTER and DIENSTEIN, 1947); calcified enamel is readily soluble in dilute acid and leaves no remnants in sections of decalcified teeth (CHASE, 1940).

Our results show that, after the full thickness of enamel is formed and latter becomes acid soluble, a new activity of the ameloblasts is evident as an accumulation of pigment in their protoplasm. This material then passes through the cuticle and is deposited into the outer zone of the enamel.

A contrary opinion, that the enamel matrix is not fully formed nor fully calcified

at the time pigmentation occurs, has been expressed by PINDBORG (1947).

According to this investigator pigmentation in rat incisors extends 3-4 mm further apically from about one-half of the total length of the lower tooth and 34 mm further apically from about two-thirds of the total length of the upper tooth. However, the pigmentation in these apical 3-4 mm does not adhere to the surface of the teeth but “can easily be wiped off”. From this macroscopic observation he concluded “that the imbedding of the pigment must take place at a time when the enamel has not yet been fully formed”.

On the basis of his histologic study (his Figs. 2-4), including the Prussian blue reaction, he believes that “in a particular place the iron leaves the ameloblasts and passes into the enamel matrix the outer part of which becomes bluish”.

Since in our specimens the yellow pigment is firmly fixed in the outer layer of enamel we cannot confirm PINDBORG’S observation that the pigmented layer could be

PIGMENTATION OF THE ENAMEL OF ALBINO RAT INCISOR TEETH 101

“easily wiped off”. Apparently he mistook the pigmented enamel organ for what he terms “pigmented outer part of enamel matrix”.

In PINDBORG’S Fig. 2, it is obvious that the form of the ameloblasts and the papillary arrangement of the remainder of the enamel organ as reproduced is inconsistent with the appearance of these structures in an area where amelogenesis is still occurring. It is also noteworthy that the zone of unstained enamel immediately below the ameloblasts is the same width as the zone of iron reacting enamel shown in his Fig. 4. The remainder of the enamel has disappeared in his Fig. 4, indicating its acid solubility. PINDBORG mistakenly interprets the acid resistance of the iron-reacting pigmented enamel shown in his Fig. 4 as indicating that it is still in the enamel matrix stage.

A positive reaction to Turnbull’s blue and Berlin blue confirms the suggestion also of other workers (DAM and GRANADOS, 1945) that this substance is an iron-rich pigment rather than a porphyrin, lipochrome or melanin. Moreover, the pigment of enamel becomes brick red upon incineration, further establishing the presence of iron. This pigment appears to harden, toughen and make acid resistant the outermost edge of rat incisor teeth.

Thus, the function of the enamel organ of the incisor tooth of the rat may be considered in three successive stages : (1) the deposition of an enamel matrix; (2) forma- tion of the enamel cuticle and (3) the mineralization of the enamel matrix. The mineralization of the matrix occurs in two phases: (a) deposition of calcium salts (maturation) and (b) deposition of iron salts with resulting pigmentation (sideration”). Since the enamel cuticle is present during the phases of maturation and pigmentation, we suggest that this membrane may play a role in the transfer of minerals from the cells of the enamel organ to the enamel matrix and calcified enamel.

Acknowledgement-Credit is due to Mr. GEORGE LEMASTER, Department of Photography, Institute of Pathology, Western Reserve University, for taking certain of the photomicrographs.

REFERENCES BOYLE, P. E. and KALNINS, V. Changes in teeth of rodents with low calcium rickets. (To be

published.)

BUTCHER, E. 0. 1953. Pigment formation in rat’s incisor. J. dent. Res. 32, 133-137. CHASE, S. W. 1940. The interrelation of maturation and the histogenesis of hypoplasia in dental

enamel. Proc. Dent. Centenary Cel. pp. 425-431. DAM, H. and GRANADOS, H. 1945. Role of unsaturated fatty acids in changes of adipose and dental

tissues in vitamin E deficiency. Science 102, 327-328.

ERDHEIM, J. 1911. Zur Kenntnis der parathyreopriven Dentinverlnderungen. Frankfurt 2. Path. 7, 238-248.

KALNINS: V. 1952. Origin of enamel drops and cementicles in the teeth of rodents. J. dent. Res. 31, 582-589.

* Since the term “maturation” has been applied to the process of calcification of enamel, the subsequent process of iron pigmentation may be called “sideration”.

102 G. STEIN AND P. E. BOYLE

NUCKOLL~, J., LEICESTER, H. M. and DIENSTEIN, B. 1947. Amelogenesis (an integration of the known factors concerned with the development, formation and mineralization of enamel). J. Amer. Coil. Dent. 14, 118-153.

PINDBORG, J. J. 1947. Studies on incisor pigmentation in relation to liver iron and blood picture in the white rat. Odont. Tidskr. 55, 443-446.

PINDBORG, J. J. 1953. The pigmentation of the rat incisor as an index of metabolic disturbances. Oral Surg. 6, 780-787.

STEIN, G. and BOYLE, P. E. 1941. Studies on enamel- I. The yellow colour of the incisor teeth of the albino rat. J. dent. Res. 20, 261 (Abstract).

TORELL, P. 1955. Iron and dental hard tissues. Odont. Tidskr. 63, 131-172.

WEINMANN, J. P. 1943. Recovery of ameloblasts. J. Amer. dent. Ass. 30, 874-888.

FIG. 1. The growth of enamel matrix occurs in the portion corresponding to 9910.45 o’clock. The tooth erupts from the alveolus at 1.30 o’clock and it appears in the mouth cavity before 2.30 o’clock (BOYLE and KALNINS).

FIGS. 2 AND 3. The relation between stages of enamel development and enamel pigmentation in upper incisors.

FIG. 2. A longitudinal unstained ground section of the tooth of a young rat. The pigment appears in the enamel about mid-way from the formative to the incisal end of the tooth; it is at a point corresponding to 12.45 o’clock.

FIG. 3. Microradiogram. Enamel is shown as a narrow radiopaque band. The arrow shows the site of initial enamel pigmentation which corresponds to 12.45 o’clock.

1

PIGMENTATION OF THE ENAMEL OF ALBINO RAT INCISOR TEETH

PLATE 1

G. STEIN AND P.E. BOYLE

PLATE 2

PIGMENTATION OF THE ENAMEL OF ALBINO RAT INCISOR TEETH

FIGS. 4 AND 5. These represent two views of an unstained ground cross section taken in the pigmented portion of an upper incisor. Fig. 4 shows the appearance before the addition of nitric acid and Fig. 5 during partial decalcification. Acid is drawn in from the left side so that enamel is further dissolved toward that side. In both figures the pigmented outer layer of enamel is shown in the form of a dark narrow band-P. Fig. 5 shows that under action of 2% nitric acid the inner non-pigmented layer of enamel becomes dissolved, whereas the outer pigmented layer (partially acid resistant) of enamel in the area indicated by X collapses toward the surface of dentine. E,-non-pigmented enamel as yet undissolved by acid. E,-space formerly occupied by the inner, non-pigmented layer of enamel. The variation between the dentine at D1 and D, is caused by a gas bubble generated during decalcification of enamel.

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104 ti. STEIN AND P.E. BOYLE

FIG. 6. The honeycomb appearance of peeled-off sheet of pigmented layer of enamel seen from the under surface view. x 500.

FIGS. 7, 8 AND 9. Show successive stages in the process of enamel pigmentation in an unstained ground section of an upper incisor in an adult rat.

In Fig. 7, upper incisor at a point corresponding to 11 o’clock, the enamel is already in full width and acid soluble. Here the ameloblasts are still without pigment. x300.

Fig. 8 shows that, at a point corresponding to 11.15 to 11.30 o’clock, accumulation of pigment in ameloblasts takes place and the cells appear yellow. x 300.

Fig. 9 demonstrates a portion of the tooth corresponding to 12-12.15 o’clock. Here the pigment disappears from the ameloblasts and becomes visible in the outer layer of enamel. x 250.

FIGS. 10 AND 11 show the enamel organ dissected away from the middle portion of the incisor, where the enamel is calcified (acid soluble) but still not pigmented. The specimen was not subjected to prior decalcification. x600. Fig. 10 shows that in the section stained with haematoxylin and eosin numerous fine granules of the yellow pigment appear in the cytoplasm of ameloblasts and in the stratum intermedium. In Fig. 11 a corresponding area is stained with Berlin blue, demonstrating that the granules give a reaction specific for iron.

PIGMENTATION OF THE ENAMEL OF ALBINO RAT INCISOR TEETH

8 IO 11

PLATE 3

PLATE 4

PIGMENTATION OF THE ENAMEL OF ALBINO RAT INCISOR TEETH

FIG. 12. Unstained longitudinal ground section showing the presence of enamel cuticle (EC) in the apical region of the incisor (at the point of 10.45 o’clock), where the enamel first becomes acid soluble. A-ameloblasts. E--enamel. x300.

FIG. 13. The sharp edge of the incisal end of a normally pigmented tooth. EP-the outer, pigmented, narrow layer of enamel. E-non-pigmented inner layer of enamel. Unstained ground section. x 500.

FIG. 14. A non-pigmented spot (indicated by an arrow) in the enamel of a left loweI incisor. This is the result of surgical destruction (by electrocauterization performed seven weeks previously) of the pigment containing enamel organ in the middle region of the tooth. Note that the colourless portion ofenamel is followed by normally pigmentedenamel. Other than the lack of colour, no defect in the enamel surface was detected. The rate of eruption of both incisors was equal.