Advances in Dental Research

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Scanning Electron Microscopy of Trypsin-Treated Enamel from Fluorosed Rat Molars T.B. Kardos, A.R. Hunter and M.J. Hubbard ADR 1989 3: 183 DOI: 10.1177/08959374890030021801 The online version of this article can be found at: http://adr.sagepub.com/content/3/2/183

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SCANNING ELECTRON MICROSCOPY OF TRYPSIN-TREATED ENAMEL FROM FLUOROSED RAT MOLARS T.B. KARDOS, A.R. HUNTER 1 , AND M J. HUBBARD 2 School of Dentistry, University of Otago, P.O Box 647, Dunedin, New Zealand; : Dental College, King Saud University, Riyadh, Saudi Arabia; and 2Department of Biochemistry, The University of Dundee, Dundee, Scotland Adv Dent Res 3(2):183-187, September, 1989 ABSTRACT

luoride-induced pitting and porosity of teeth have long been observed, but little progress has been F made in determining their origin. We have observed, in the trypsin-treated surfaces of enamel, pits that disappear on completion of maturation, following the removal of the protein matrix and full mineralization. Since these pits were considered to be similar to those seen in fluorotic teeth, this scanning electron microscope (SEM) study was undertaken to determine the effect of fluoride on these transient developmental pits during enamel matrix maturation. A group of 20 eight-day-old rats was given daily intraperitoneal injections of NaF (20 mg/kg [9 mg F~/kg] body weight) for five days. Twenty control animals received intraperitoneal injections of isotonic saline. Maxillary and mandibular molars were dissected from the 13-day-old animals, washed in HEPES buffered (Ca2+/Mg2+) free basal medium, Eagle's (BME), incubated in 3% trypsin/BME for 5-10 min at room temperature, then indirectly sonicated in BME for 2-4 min. Clean crowns were fixed in 2.5% glutaraldehyde for three hr, dehydrated, critical-point-dried, and sputter-coated for SEM. Pits in the surfaces of developing enamel were observed in all groups. In control teeth, the pitting was restricted to the cervical margin, whereas in teeth from the fluoride-treated animals, pits were observed on some cuspal surfaces in addition to the cervical margin. These results confirmed that pits in trypsin-treated surfaces of developing enamel are a transient developmental event and showed that, in the presence of a high dose of fluoride, the maturation of enamel is modified with retention of the pits.

INTRODUCTION Dental fluorosis manifests itself as a range of clinical appearances and, in severe forms, is characterized by enamel opacities and discrete pits that may often be confluent (Dean, 1934). Although the clinical manifestations have been extensively described, few studies have been systematically aimed at determining the origins of the pits (Fejerskov et al., 1977). Differing hypotheses have been proposed over whether the lesions are developmental or caused by postPresented at the Symposium and Workshop on Developmental Defects of Enamel, February 23-25, 1988, Rotorua, New Zealand, sponsored by Colgate-Palmolive (NZ) Ltd., the New Zealand Dental Research Foundation, and the Medical Research Council of New Zealand This investigation was supported in part by a grant from the Medical Research Council of New Zealand.

emergence events. They are generally considered to be the result of a hypoplastic process (Suckling and Purdell-Lewis, 1982), due to some dysfunction of the ameloblasts, although others have suggested that they arise from failure of secondary calcification (Thylstrup and Fejerskov, 1979). In contrast to other mineralized tissues, enamel matrix undergoes mineralization as it is secreted with crystals approximating the secretory ameloblasts. As the matrix matures, differences in histochemical staining between the recently secreted matrix and a deeper zone are evident (Eastoe and Camilleri, 1971). Eastoe and Camilleri interpreted their results as demonstrating protein-rich and mineral-rich zones, although the presence of such zones is not supported by ultrastructural investigations (Frank and Nalbandian, 1967). The general morphology of fluorosed enamel, as observed by light and transmission electron microscopy, does not differ greatly from normal enamel 183


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(Fejerskov et al., 1977), although one of the characteristics of fluorosed enamel is an increase in porosity resulting from the prisms being surrounded by large crystal-free gaps (Fejerskov et al., 1974). Previous scanning electron microscope (SEM) investigations of developing enamel (Boyde, 1967; Skobe, 1967, 1977; Thylstrup et al., 1977) have utilized specimens that have been fixed prior to the removal of organic debris. However, since chemical fixation cross-links proteins that may mask the surface of the incompletely mineralized matrix, we have developed a procedure of removing the organic material prior to fixation, a preliminary report of which has appeared earlier (Hubbard and Kardos, 1982). Since developmental pits, similar to those of fluorotic enamel, were observed in the trypsin-treated surface during maturation of normal enamel, we investigated the effect of a high fluoride dose on the trypsin-treated surfaces of rat molar tooth germs.

Fig. 1 — Occlusal view of a control mandibular first molar, showing smooth contours extending from the cusp tips to the cervical margin, indicating complete maturation of enamel. Bar = 1 mm.

MATERIALS AND METHODS

We used two groups of eight-day-old mixed-sex Wistar rats from nine litters. Each litter provided randomly selected experimental and control animals. One group of 20 weanlings was given a daily intraperitoneal injection of NaF, 20 mg/kg body weight (9 mg F" per kg body weight). A control group of 20 animals received daily intraperitoneal injections of sterile isotonic saline. The volume given to each of the control animals was calculated to be the same as would have been given to an experimental animal of comparable weight. Animals in both groups were dosed for five consecutive days and were returned to their mothers' cages after being dosed. At 13 days of age, the animals were killed and the maxillary and mandibular first and second molars rapidly dissected (Kardos and Hubbard, 1981). Crowns were washed in HEPES buffered (Ca2+/Mg2+) free basal medium, Eagle's (BME), with L-glutamine, pH 7.4, incubated for five min in 3% trypsin/BME at room temperature, then transferred to fresh BME and indirectly sonicated in a water bath for two min. Specimens were examined under a dissecting microscope so that we could determine if adherent soft tissues remained, and if necessary, the trypsinization/sonication procedure was repeated. Cleaned crowns were fixed for three hr in 2.5% glutaraldehyde in 0.1 mol/ L cacodylate buffer, pH 7.3, wrapped individually in gauze, dehydrated, and critical-point-dried. Specimens were mounted onto stubs with double-sided adhesive tape, sputter-coated with gold, and examined in a Siemens ETEC scanning electron microscope operating at 20 kV. RESULTS

Enamel maturation, assessed by smooth contours of the enamel surface, was almost complete in control

Fig. 2 —Lateral view of a control mandibular first molar, showing smooth enamel contours extending from the cusp tips to the cervical margin. Several scattered pits (arrowheads) are evident in the trypsin-treated enamel in the region of the cervical margin, indicating that maturation of this region is not complete. Bar = 1 mm.

first molar crowns, with smooth surfaces extending from the cusp tips to the cervical margin region, where a few scattered pits were evident (Figs. 1 and 2). Maturation was slightly more advanced in the mandibular teeth, since the extent of cervical pitting was less than that seen in the maxillary teeth. Further, most of the first molars showed evidence of root development, indicating that eruption had commenced. In the second molars, the smooth contours of enamel were established on the cuspal planes, but a broad band of presumably incompletely mineralized enamel with numerous pits was evident near the cervical margins (Fig. 3).


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Fig. 3 —Oblique view of a control mandibular second molar, showing prominent pits in the enamel of the cervical region of the crown. The pits are more prominent on the distal surface. Bar = 1 mm.

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Fig. 5 —Occlusal view of experimental mandibular first molar, from an animal that was dosed with fluoride, showing the retention of numerous pits in the enamel on the cuspal planes. Comparison with a control tooth (Fig. 1) shows that maturation of enamel matrix is delayed. Bar = 1 mm.

DISCUSSION

Fig. 4 —Latero-occlusal view of a mandibular second molar, from an animal that was dosed with fluoride, showing numerous pits in the enamel of the cuspal planes and in the cervical region. The smooth contours of mature enamel matrix shown in the control teeth (Fig. 2) have not been established. Bar = 1 mm.

In contrast to the control teeth, the pits in the surfaces of fluoride-treated teeth persisted; the smooth contours of enamel, as seen in the cuspal and cervical areas of control teeth, were not established. Scattered pits of variable size were evident on most surfaces of both first and second molars (Figs. 4 and 5). No quantification of the distribution of the pits was attempted, but they appeared to be in greater numbers on the maxillary than the mandibular teeth. Although the pits showed variations in distribution, size, and morphology, they were similar to those seen in some control teeth, particularly those seen on second molars.

Developing enamel is rich in organic matrix proteins that dramatically decrease as the tissue matures. Although it is generally accepted that the mineralization of enamel is matrix-directed, the mechanism by which this occurs is not known, in spite of the abundance of morphological and biochemical data (Lyaruu, 1986). Scanning electron microscopy is a valuable adjunct to histological and ultrastructural techniques for studies of developing enamel, since entire surfaces can be visualized and three-dimensional structural relationships determined. In an SEM study of molar tooth germs of mice, we observed the trypsin-treated surface of maturing dental enamel and demonstrated the presence of numerous scattered pits of variable size. These pits were surrounded by regions of trypsin-resistant enamel, and they were observed on all surfaces of the molar teeth from early stages of enamel maturation. Their numbers decreased as maturation progressed, reflecting previously established cuspocervical and mesio-distal maturation gradients (Weinmann et ah, 1942). However, molars from animals of the same age that were fixed, prior to removal of the organic debris (Boyde, 1967), showed smooth contours to the enamel surface at all similar stages of matrix maturation. The different contours of the enamel surfaces seen by the two methods can be explained by the changes in physical properties of organic matrices that occur during fixation (Kardos and Simpson, 1979) and the resultant preservation of the developing enamel surface. Removal of a presumably protein-rich irregular surface region of enamel by enzyme treatment and sonication prior to fixation, how-


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ever, revealed the configuration of a deeper region of presumably more mineralized enamel. Angmar-M&nsson and Whitford (1984) have considered rats to be a suitable model for the study of human fluorosis. This conclusion was based upon their findings that identical plasma concentrations of fluoride, in the two species, resulted in comparable enamel defects, and upon the observations of Warshawsky et al. (1981) that there is a common similarity in the basic structure and mode of enamel formation. The present study was undertaken on rat molars, since the enamel organ of these teeth has been reported to be more sensitive to fluoride than the incisor (Mornstad and Hammarstrom, 1978), and since enamel production occurs for a limited period, with maturation of the matrix progressing cuspo-cervically. Variations in the maturation of the enamel matrix were shown to occur between teeth and surfaces in animals of the same strain. Generally, mineralization of the molar crowns in rats commences shortly after birth and is complete by 13 days of age, for first molars just prior to eruption, with mineralization and maturation of the enamel matrix occurring between 8 and 13 days (Cheyne, 1942). Since fluoride was administered over the period of enamel matrix maturation and comparisons made with control teeth of similar chronological age, the retention of the developmental pits could be attributed to modifications of the maturation process in the presence of fluoride. Further, our results support earlier suggestions that many defects of enamel are manifestations of normal developmental events (Francis and Briner, 1966; Newman and Poole, 1974). The maturation of enamel matrix could have been modified as a result of changes in cellular activity (Suckling and Purdell-Lewis, 1982; Newman and Poole, 1974), secondary crystal growth (Kruger, 1970; Takuma et al., 1984), proteolysis of the matrix (Hammarstrom, 1971), rheological properties of the matrix (Fejerskov et al., 1977), or any combination of the above. The distribution of the pitting and the sizes of pits were variable between teeth within the experimental groups, but their presence and morphology were a consistent finding. Some variations within each group can be accounted for by differences in metabolic activity of ameloblasts. The susceptibility of ameloblasts to fluoride (Kruger, 1968, 1970; Walton and Eisenmann, 1974; Takuma et al., 1984; Ashrafi et al., 1987) and their ability to recover can be related to a particular stage in their life cycle. In response to injury, secretory ameloblasts recover rapidly to produce a layer of normal enamel over hypoplastic areas (Boyde, 1970; Takuma et al., 1984), whereas cells in the transitional stage are particularly sensitive and may not recover (Josephsen and Fejerskov, 1977; Shinoda and Ogura, 1978; Takuma et al., 1984). Since the period of rapid ion influx into the matrix commences at the transitional stage (Robinson and Kirkham, 1985), it is likely that fluoride dosing coincided with the time that the ameloblasts were particularly susceptible to

changes in their environment (Kardos and Hubbard, 1984). Further study is necessary, however, to determine if the retention of the developmental pits, seen in the present study, is a result of the failure of ameloblasts to enter the maturation phase. The developmental pits shown in this study are unlikely to be restricted to rodent teeth. Similar structures, containing organic-rich material, have been demonstrated in an SEM study of human teeth (Newman and Poole, 1974). They may represent large round crystal-free spaces deep in enamel that contain cellular debris (Takuma et al., 1984), into which crystals grow as the normal maturation of enamel continues to completion. Since we have shown that these developmental pits persist in the presence of fluoride, it is reasonable to conclude that modification of enamel maturation is a primary factor in the pathogenesis of enamel fluorosis (Shinoda, 1975; Fejerskov et al., 1977). Modifications to the normal development of enamel can account for the high prevalence (from 30-50%) of "fluorotic enamel" in populations (Cutress and Suckling, 1982) and the similarities in the distribution and extent of porous areas in fluorotic and normal enamel (Fejerskov et al., 1977). Although fluoride is implicated as the etiological factor for many enamel defects, similar lesions may occur in the absence of fluoride as a result of the action of a number of vastly different factors (Kreshover, 1960; Cutress and Suckling, 1982). It is likely that these factors also modify the process of enamel maturation; hence, similar lesions may arise from vastly different etiological factors. The retention of pits in the enamel surface suggests that organic-rich areas are likely to be present in enamel at the time of eruption. Consequently, teeth may emerge with an intact surface, but latent pits beneath the surface may be exposed with function (Francis and Briner, 1966; Thylstrup and Fejerskov, 1977; Takuma et al., 1984). REFERENCES ANGMAR-MANSSON, B. and WHITFORD, G.M. (1984): Enamel Fluorosis Related to Plasma F~ Levels in the Rat, Caries Res 18:25-32. ASHRAFI, S.H.; EISENMANN, D.R.; and ZAKI, A.E. (1987): Secretory Ameloblasts and Calcium Distribution during Normal and Experimentally Altered Mineralization, Scanning Microsc 1:1949-1962. BOYDE, A. (1967): The Development of Enamel Structure, Proc R Soc Med 60:923-928. BOYDE, A. (1970): The Surface of the Enamel in Human Hypoplastic Teeth, Arch Oral Biol 15:897-898. CHEYNE, V.D. (1942): Production of Graded Mottling in Molar Teeth of Rats by Feeding Potassium Fluoride, / Dent Res 21:145155. CUTRESS, T.W. and SUCKLING, G.W. (1982): Non-carious Defects of Enamel: Types, Occurrence and Classification, Int Dent J 32:117-122. DEAN, H.T. (1934): Classification of Mottled Enamel Diagnosis, / Am Dent Assoc 21:1421-1426. EASTOE, J.E. and CAMILLERI, G.E. (1971): The Pattern of Protein Distribution of Enamel During Maturation. In: Tooth Enamel


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SHINODA, H. and OGURA, H. (1978): Scanning Electron Microscopical Study of Fluorosis of Enamel in Rats, Calcif Tissue Res 25:75-83. SKOBE, Z. (1976): The Secretory Stage of Amelogenesis in Rat Mandibular Incisor Teeth Observed by Scanning Electron Microscopy, Calcif Tissue Res 21:83-103. SKOBE, Z. (1977): Enamel Rod Formation in the Monkey Observed by Scanning Electron Microscopy, Anat Rec 187:329-334. SUCKLING, G.W. and PURDELL-LEWIS, D. (1982): Macroscopic Appearance, Microhardness and Microradiographic Characteristics of Experimentally Produced Fluorotic Lesions in Sheep Enamel, Caries Res 16:227-234. TAKUMA, S.; FURUMI, A.; TOMODA, F.; OGIWARA, H.; KUMAMOTO, Y.; and YANAGISAWA, T. (1984): Ultrastructural Studies of Disturbances in Amelogenesis Induced in Rat Incisors by Fluoride and Strontium Administration. In: Mechanisms of Tooth Enamel Formation, S. Suga, Ed., Tokyo: Quintessence Publishing Co., Inc., pp. 259-272. THYLSTRUP, A. and FEJERSKOV, O. (1979): A Scanning Electron Microscopic and Microradiographic Study of Pits in Fluorosed Human Enamel, Scand } Dent Res 87:105-114. THYLSTRUP, A.; SKAARING, P.; FEJERSKOV, O.; and BIERRING, F. (1977): Surface Structure of Tooth Germs from New Born Infants, a Light and Scanning Electron Microscopical Study, / Anat 123:537-547. WALTON, R.E. and EISENMANN, D.R. (1974): Ultrastructural Examination of Various Stages of Amelogenesis in the Rat Following Parental Fluoride Administration, Arch Oral Biol 19:171182. WARSHAWSKY, H.; JOSEPHSEN, K.; THYLSTRUP, A.; and FEJERSKOV, O. (1981): The Development of Enamel Structure in Rat Incisors as Compared to the Teeth of Monkey and Man, Anat Rec 200:371-399. WEINMANN, J.P.; WESINGER, G.D.; and REED, G. (1942): Correlation of Chemical and Histological Investigation of Developing Enamel, / Dent Res 21:171-182.
