The Effect of Strontium, Cobalt and Fluoride on R a t Incisor Enamel Formation ' B R A H A M NEIMAN AND DALE R. EISENMANN The Department of Histology, University of Illinois at the Medical Center, Chicago, Illinois 60680
This investigation examined ultrastructurally the entire period of development of alterations in formative ameloblasts and the enamel which they produce following injection with fluoride, strontium, and cobalt ions. Rats injected with these ions were sacrificed at intervals of 1, 2, 4, 8, 16,24 and 48 hours to elucidate the sequence and detail of cytologic and cell product alterations which occur. Undecalcified sections of rat incisor teeth were studied using electron microscopy and microradiography. All three ions initially produced disturbances in cell morphology and enamel formation consisting of dark globules, vacuoles, and pooling of stippled material on the enamel surface. While a period of decreased crystal formation occurred after injection with all three ions, only cobalt responses included a period of apparently complete absence of crystd formation. The hypermineralized layers occurring in the altered enamel are attributed to changes in the rate of enamel matrix formation and duration of its exposure to tissue fluids. Morphologic changes in Tomes' process were observed at the time of formation of abnormal enamel following injection of all three ions. These observations are compared with previous studies of altered enamel formation and analyzed with the goal of learning more about the mechanisms of amelogenesis. ABSTRACT
Schour and Smith, '34, '35) showed that subcutaneous injections of sodium fluoride caused disturbances in enamel formation as layers of hypomineralized and hypermineralized enamel. They observed large darkly stained globules in the ameloblast a few hours following injection. Several light and electron microscopic studies since then have also reported morphological changes in the formative ameloblast induced by fluoride (Bhussry, '59; Kruger, '67, '68, '70; Walton and Eisenmann, '72). These changes consistently include large, darkly stained globules and large vesicles in the distal region of the cell body. Weinmann ('43) reported that injected strontium caused enamel hypoplasia and hypomineralization. The ameloblasts were seen to undergo degenerative changes and detachment from the enamel matrix. Recent electron microprobe studies of the effect of dietary strontium on forming enamel revealed a n increased uptake of strontium and decreased uptake of calcium and phosphate (Johnson and Singer, '67). ANAT.REc., 183: 303-322.
Microradiography has been used to assess mineralization disturbances induced by fluoride, strontium, and cobalt (Allen, '63; Weber and Yaeger, '64; Yaeger, '66; Eisenmann and Yaeger, '69; Kruger, '71). Fluoride produced a double response i n rat enamel consisting of a n inner (first formed ) hypermineralized layer followed by a n outer layer. Cobalt produced first a hypomineralized layer and then a hypermineralized layer, the inverse sequence of fluoride. Strontium produced a hypermineralized enamel followed by a diffuse continuous hypomineralized area. Cell damage has been suggested as being responsible for all developmental mineralization lesions except those caused by amdogenesis imperfecta (Nylen et al., '72). Through electron microscopy, disturbances in rat enamel induced by injections of tetracycline were shown to result from a change in rate of matrix secretion Received Aug. 22, '74. Accepted Apr. 3, '75. 1This study was supported in part by grant PHS DE03312 from the National Institute for Dental Research.
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and disturbances in nucleation. The inner hypermineralized layer was thought to be caused by uninterrupted ion transport to previously nucleated crystals during the period when the hypomineralized layer was formed. Disturbances in crystal growth were attributed to the inability of the injured secretory ameloblast to fully recover and function normally during its maturative phase. Previously cited studies have examined alterations in enamel and changes in morphology of cells following various inducing agents. These studies were limited in that they usually considered these disturbances only after they were completed. Investigators have usually studied either the alterations of cellular morphology or variations in enamel density, but with little correlation of these two aspects. This study examines the ameloblasts and their product during the entire period of apposition of altered enamel, utilizing parenteral dosages of strontium, cobalt, and fluoride known to induce disturbances in enamel formation (Eisenmann and Yaeger, '69). Its purpose is to gain further insight into some of the mechanisms of amelogenesis. MATERIALS AND METHODS
Electron microscopy Male Sprague-Dawley rats weighing 175-250 g were given a single subcutaneous injection of either 5 m g sodium fluoride, 125 mg strontium chloride (Yaeger and Eisenmann, '63), or 100 m g cobalt chloride/100 g body weight (Eisenmann and Yaeger, '69) and sacrificed at intervals of 1, 2, 4, 8, 16, 24 and 48 hours. A minimum of two animals was utilized for each time interval. This time period covers the development of the altered enamel produced by the secretory ameloblasts. Control animals were given identical dosages of sodium chloride and were sacrificed at 1, 2 and 4 hours. Our control animals plus controls done in a previous study (Eisenmann and Yaeger, '69) at longer time intervals confirm that the enamel response is not a result of the injection procedure itself. The rats were anesthetized with ether and fixed by vital perfusion through the aorta with 4% gluteraldehyde in 0.04 M cacodylate buffer
at pH 7.2. The maxillary incisors were reremoved along with the surrounding alveolar bone keeping the enamel organ intact. [ncisors were mounted on a Gillings-Hamco sectioning machine and longitudinal sections of 150-300 were cut through the middle third using normal saline solution as a coolant. These sections were returned to cold gluteraldehyde for one hour, rinsed in cacodylate buffer for one hour, and post-fixed in veronal buffered 1% osmium tetraoxide for one hour. Specimens were then dehydrated in a graded series of alcohols and flat-embedded in araldite for cross-sectioning. Sectioning was one with a Porter-Blum microtome equipped with a diamond knife. The sections were taken from the region where inner enamel had reached a thickness of 20-50 pm. Thick sections (0.5 pm) were examined in the light microscope to assist in achieving the proper location and orientation. Ultra-thin sections were floated on a saturated solution of dibasic calcium phosphate (Boothroyd, '64), collected on parloidin and carbon-coated grids and stained with uranyl magnesium acetate and lead citrate. The specimens were examined with a Hitachi 7-S electron microscope at 50 kv.
Microradiography Forty-eight hour specimens of cobalt, strontium, and fluoride were embedded in methyl methacrylate. Two week specimens of strontium were similarly processed. Longitudinal sections of 80-100 pm in thickness were cut from the middle third of the tooth with a Gillings-Hamco sectioning machine. Microradiographs were obtained on Kodak 649-0 spectroscopic plates using a nickel filter in a Siemens x-ray generator operated at 19 kV. OBSERVATIONS
Electron microscopy The typical morphology of a tall columnar ameloblast equipped with abundant organelles for protein synthesis and secretion were observed i n all control specimens (fig. 1). 1. Fluoride The morphology of the ameloblast maintains a normal appearance one hour after
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injection with fluoride. After two hours numerous large vacuoles and dark globules appear to be stacked-up in the distal half of the cell body (fig. 2). These spherical bodies vary in size and density and are often large enough to span nearly the entire width of the cell, displacing profiles of endoplasmic reticulum. The bodies are membrane-bound and do not appear to communicate with each other or with any other organelle. The organelles of the cell body are of a normal appearance and the quantity of secretion granules is similar to that of the control cells. No alterations appear in Tomes’ process or the adjacent enamel crystals a t this stage of the response to fluoride. After four hours, the dark globules and vacuoles increase in number and in some cases extend to the nuclear region. Small pools of stippled material accumulate just outside of the distal end of Tomes’ process. Other than the large bodies, the general appearance of the components of the cell body and Tomes’ process appear unaltered four hours after fluoride. At eight hours, larger lakes of stippled material form a t the distal ends of Tomes’ processes (fig. 3). There is a decrease in enamel formation between the processes. The large bodies seen earlier in the cell body have decreased in number and size. At 16 hours the cells appear to have nearly recovered from the effects of fluoride. The dark globules and vacuoles seen i n the central region of the ameloblasts are similar in size and amount to those seen i n the control cells. No alterations are present in any of the organelles, but the morphology and size of Tomes’ process has been altered. Enamel formation has resumed between the Tomes’ processes and a t the distal end of each process leaving behind large lakes of noncrystallized or sparsely crystallized stippled material. At 24 and 48 hours after the injection the ultrastructure of the ameloblast shows no unique features other than a continued alteration after 24 hours in Tomes’ process (fig. 4 ) . Normal enamel is being laid down over the hypomineralized band. The layer of altered enamel consists of areas containing scattered disoriented crystals in a homogeneous osmiophilic background and spaces devoid of mineral but contain-
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ing a loosely arranged granular material (fig. 5). 2. Ctjbalt The ameloblast maintains a normal appearance two hours after injection with cobalt. Small pools of stippled material lacking crystals accumulate just outside of the distal and mid-proximal regions of Tomes’ process. At four hours after injection, numerous large vacuoles and dark globules varying in size and density appear in the distal third of the cell body (fig. 6). The quantity and size of the spherical bodies are similar to those seen after injection of fluoride. After eight hours the large spherical bodies seem to have decreased in size and number. The morphology and size of Tomes’ process is altered. Lakes of stippled materisl accumulate a t the distal end of Tomes’ process (fig. 7). At 24 and 48 hours after the injection, the ultrastructure of the ameloblast appears normal. Crystal formation occurs after 24 hours over the altered hypomineralized regions leaving behind lakes of non-crystalline stippled material (fig. 8). A resumption of normal enamel formation occurs overlying the hypomineralized region (fig. 9 ) . The thickness of enamel formed over the hypomineralized region is less than the amount formed after the fluoride response (fig. 5).
Strontium One and two hours after injection with strontium, the ultrastructure of the ameloblast reflects no morphological differences from control cells. After four and eight hours, large vacuoles and dark globules accumulate in the distal half of the cell body (fig. 10). Small lakes of stippled material pool at the distal end of Tomes’ process and along the enamel spikes. At eight hours, the enamel front exhibits a disorganization of crystals and a lack of prism morphology. At 16 hours, the spherical bodies decrease in size and amount. Observations of the organelles reveal nothing abnormal. Pools of stippled material accumulate in the intercellular spaces between the processes along with a decrease in the formation of interprismatic enamel in these 3.
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areas, A broad spectrum of alteration is noted in the size and morphology of the Tomes’ process of some cells. The normal prismatic pattern of enamel is not evident in such areas. At 24 hours, large lakes of stippled material accumulate on the enamel surface and more proximally between the Tomes’ processes. Large vacuoles and dense globules again appear in the distal cytoplasm. Tomes’ processes are severely altered in size and morphology and the adjacent enamel contains scattered, disorganized crystals. At 48 hours after injection, numerous large vacuoles and dark globules varying in size and density persist in the distal half of the cells (fig. 11). The distal end shows a reduction of Tomes’ process into thin finger-like structures of varied shapes. Huge lakes of non-crystalline, dense homogeneous material accumulate in the intercellular spaces between ameloblasts on the surface of the enamel (fig. 12). The mineralizing front contains a decreased concentration of crystals which are haphazardly organized (fig. 11). This area represents formation of a second hypomineralized layer. On either side of the first-formed hypomineralized band, there is a lack of prism morphology. The organic matrix observed in the first-formed hypomineralized regions and that which is accumulating at the enamel front consists of a n osmiophilic material containing a few scattered crystals.
Microradiograp hy Following are microradiographic descriptions of the response to each ion at 48 hours after injections in the formative stage of amelogenesis : Fluoride. The response to fluoride consists of two components: a n inner (firstformed) hypermineralized band, and a n outer hypomineralized component covered with a thin layer of newly formed normal enamel (fig. 13). Cobalt. The response to cobalt consists of a n inner hypomineralized region which precedes a n outer hypermineralized layer (fig. 4). On the surface is a layer of newly formed normal enamel. Strontium. The response seen in the microradiograph consists of up to four
components in some regions. Proceeding from the inner (first-formed) layer outward they are : 1. a n inner hypennineralized layer; 2. a hypomineralized layer; 3. a hypermineralized layer; 4. a n outer hypomineralized region, Little, if any, normal enamel is formed external to the outer hypomineralized layer (fig. 15). An additional %week follow-up examination of the strontium response revealed a complete lack of resumption of normal enamel secretion in the affected region with normal enamel formation by ameloblasts developing later (fig. 16). DISCUSSION
Electron microscopic investigation of amelogenesis disturbed by strontium, fluoride, and cobalt has revealed alterations i n both cell morphology and enamel formation. Early changes following injection with all three ions consist of accumulations of dark globules and vacuoles in the cell and pooling of stippled material on the enamel surface. Permanent recovery of enamel formation occurs after cobalt and fluoride, but the brief recovery by the ameloblasts after injection with strontium is followed by another severe response in the cell and enamel after 48 hours and a lack of subsequent recovery in the affected area. Johnson et al. (’68) have demonstrated that strontium incorporated in the bone of rats is subsequently translocated to the incisor teeth during the remodeling process of bone. This process may be responsible for the second wave of disturbance following strontium injection. The orientation of enamel crystals and the resultant formation of a highly ordered prismatic structure has been attributed to the organizing properties of the Tomes’ process cell membrane (Boyde, ’64). Variations in Tomes’ process were observed in ameloblasts at the time of formation of abnormal enamel following injection of all three ions. The severest alteration of Tomes’ process was seen after injection with strontium which also produced the most extreme changes in enamel structure. The least severe disturbance of Tomes’ process was produced by cobalt which induced the mildest defect insofar as prism morphology is concerned. Variations in enamel structure are also seen during nor-
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ma1 development when formation has first begun at the dentino-enamel junction and when it is nearly completed producing layers of prismless enamel. At both of these stages of enamel formation Tomes' processes are undergoing marked changes in morphology : either developing as cell processes or being phased out. This information along with the currently observed relationship between Tomes' process morphology and the quality of enamel structure, support an organizational function for Tomes' process. A number of factors must be considered in analyzing mechanisms of interference with enamel formation. Among these are quality of matrix, crystal nucleation and growth, transfer of mineral ions to the matrix and systems acting as regulators of mineralization. Although it has been demonstrated autoradiographically (Kruger, '70) that some variation in enamel matrix occurs following fluoride injection, poorly mineralized areas OE dentin induced by strontium and fluoride were found capable of accumulating mineral in vitro (Eisenmann and Yaeger, '72). This indicates that the organic matrix, if it was altered, was at least not permanently resistant to mineralization. The pools of unmineralized stippled material observed in the present study as well as the regions of sparsely mineralized matrix are present at times when mineral is being deposited in adjacent hypermineralized zones of the strontium and fluoride responses. The resistance to mineralization by specific regions may be due to localized interference with certain of the other factors involved in the mineralization process and known to be susceptible to interference by at least some of the ions injected. For example, enzyme systems essential for proper cell function are inhibited by fluoride (Frajola, '59; Yoshida et al., '68); and cobalt, fluoride and strontium are reported to have various inhibitory effects on crystal nucleation and growth (Bird and Thomas, '63). In addition, it has been suggested by Russell and Fleisch ('70) that fluoride may exert its effect on mineralization indirectly by inhibiting phosphatase and permitting a greater action by pyrophosphate, a known inhibitor of mineralization.
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Recent evidence points toward the active involvement of formative cells in transferring mineral ions from the circulating fluids to various mineralizing tissues (Kashiwa and Mukai, '71; Martin and Matthews, '70; Kuhar, '74). If the ameloblast is a mineral transporting cell, it is conceivable that the injected ions could interfere with this process by enzyme disturbances, membrane alterations, or by calcium and phosphate binding within the cell. Fluoride-induced enamel changes appear to consist of localized interference with mineralization of newly formed matrix with concurrent accumulation of available calcium and phosphate ions in the preresponse enamel. This pattern of formation of hypermineralized enamel has been proposed by previous authors (Weber and Yaeger, '64; Nylen et al., '72). Only isolated areas of matrix in the hypomineralized layer of some specimens appear to be so severely altered morphologically as to prevent mineralization completely. Those areas of matrix which do contain crystals but of a deficient number appear otherwise similar to normal enamel matrix. Thus it is likely that mineral ion flow has continued and a brief localized alteration in mineralization of newly forming matrix has occurred. This may be a result of inhibition of enzymes such as phosphatase and/or interference by fluoride with the processes of crystal nucleation and growth. Complete recovery of the ameloblast is indicated by the substantial thickness of normal enamel deposited over the hypomineralized layer. The response to strontium may be considered as a double fluoride response. Both ameloblastic and enamel alterations g o through two cycles which, as mentioned previously, may reflect a secondary wave of strontium ions as they are released from bone, Strontium eventually leads to a cessation of enamel formation which is evidenced by the lack of post-response enamel observed microradiographically two weeks after injection. The matrix produced shortly after cobalt injection remains almost completely devoid of crystals. It is interesting to note that the appearance of this unmineralized ma-
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trix is no different from that of normal stippled material. Also it is significant that no hypermineralization occurs in the preresponse enamel. These observations lend support for speculation that a temporary interruption in mineral ion flow may occur. However, it seems unlikely that this alone could be responsible for the hypomineralized layer, because when ion flow is resumed it would lead to crystal formation in the older crystal deficient regions as well as the newly formed matrix. As a known inhibitor of nucleation (Bird and Thomas, '63), cobalt may be present in the hypomineralized region and thus prevent its mineralization even after ion flow resumes. The formation of a subsequent hypermineralized layer may well be due to a period of decreased rate of enamel matrix formation as evidenced by the lesser amount of post-response enamel as compared to after fluoride. Ion induced disturbances i n amelogenesis lead to various combinations of cellular and enamel defects which have many aspects in common along with certain major variations. Nylen et al. ('72) have proposed that most disturbances in ameloblast function follow a similar pattern one that is comparable to that observed here for fluoride and strontium. However, i t appears that the response to cobalt is unique and may lend itself to further study of some of the intricate mechanisms by which normal enamel is formed. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Mrs. Elena Baltrusaitis for her technical assistance and Mrs. Izabele Stoncius for her photographic work. LITERATURE CITED Allan, J. H. 1963 Observations o n the development of dental enamel in acute experimental fluorosis. In: Advances in Fluorine Research and Dental Caries Prevention. J. L. Hardwick, J . P. Destin and H. R. Held, eds. The Macmillan Co., New York, pp. 41-51. Bhussry, B. R. 1959 Effects of sodium fluoride on the developing teeth of rats. J. Dent. Res., 38: 653-654. Bird, E. D., and W. C. Thomas, Jr. 1963 Effect of various metals on mineralization in vitro. Proc. SOC.Exp. Biol. Med., 112: 640-643. Boothroyd, B. 1964 The problem of demineral-
ization in thin sections of fully calcified bone. J. Cell Biol., 20: 165-175. Boyde, A. 1964 The Structure and Development of Mammalian Enamel. Ph.D. Thesis, London Hospital Medical College, London. Eisenmann, D. R., and J. A. Yaeger 1969 Alterations in the formation of rat dentin and enamel induced by various ions. Archs. Oral Biol., 1 4 : 1045-1064. 1972 In vitro mineralization of hypomineralized dentin induced by strontium and fluoride. Archs. Oral Biol., 27: 987-999. Frajola, W. J. 1959 Fluoride and enzyme inhibition. In: Fluorine and Dental Health. The Pharmacology and Toxicology of Fluorine. J. C. Muhler and M. K. Hine, eds. Indiana University Press, Bloomington, pp. 68-69. Johnson, A. R., W. D. Armstrong and L. Singer 1968 The incorporation and removal of large amounts of strontium by physiologic mechanisms ni mineralized tissues of the rat. Calc. Tiss. Res., 2: 242-252. Johnson, A. R., and L. Singer 1967 A n electronmicroprobe study of rat incisor teeth with low or high concentrations of strontium. Archs. Oral Biol., 12: 389-399. Kashiwa, H. K., and C. D. Muai 1971 Lipidcalcium-phosphate spherule i n chondrocytes of developing long bones. Clin. Orth., 78: 223-229. Kruger, B. J. 1967 Histologic effects of fluoride and molybdenum on developing dental tissues. Aust. Dent. J., 12: 54-60. 1968 Ultrastructural changes in ameloblasts from fluoride treated rats. Archs. Oral Biol., 13: 969-977. 1970 An autoradiographic assessment of the effect of fluoride on the uptake of H3-proline by ameloblasts in the rat. Archs. Oral Biol., 15: 103-108. 1970 The effect of different levels of fluoride on the ultrastructure of ameloblasts in rat. Archs. Oral Biol., 15: 109-114. 1971 Scanning electron microscopy of sections of fluorosed rat enamel. J. Dent. Res., 50: 1685. Kuhar, K. J. 1974 Fluoride-induced Calcification within the Maturative Ameloblast of the Rat. M. S. Thesis, University of Illinois, Chicago. Martin, J. H., and J. L. Matthews 1970 Mitochondrial granules in chondrocytes, osteoblasts and osteocytes. Clin. Orth., 68: 273-278. Nylen, M., K. Ommell and C. Lofgren 1972 A n electron microscopic study of tetracyclineinduced enamel defects in rat incisor enamel. Scand. J. Dent. Res., 80: 384-409. Russell, R. G., and H. Fleisch 1970 Inorganic pyrophosphate and pyrophosphatases in calcification and calcium homeostasis. Clin. Orth., 69: 101-117. Schour, I., and M. C. Smith 1934 The histologic changes in the enamel and dentin of the rat incisor i n acute and chronic experimental fluorosis. Univ. Ariz. Coll. Agric., Agric. Exp. Sta., Techn. Bull., 52: 67-91. 1935 Mottled teeth: An experimental histologic analysis. J. Am. Dent. Assoc., 22: 796-8 13.
ION INDUCED DISTURBANCES IN AMELOGENESIS Walton, R. E., and D. R. Eisenmann 1974 Ultrastructural examination of various stages of amelogenesis in the rat following parental fluoride administration. Archs. Oral Biol., 19: 171-183. Weber, D. F., and J. A. Yaeger 1964 A microradiographic interpretation of abnormal enamel formation induced by subcutaneous sodium fluoride. J. Dent. Res., 43: 50-56. Weinmann, J. P. 1943 Recovery of ameloblasts. J. Amer. Dent. Assoc., 30: 874-888. Yaeger, J. A. 1966 The effects of high fluoride
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diets on developing enamel and dentin in the incisors of rats. Am. J. Anat., 18: 665-683. Yaeger, J. A,, and D. R. Eisenmann 1963 Response in rat incisor dentin to injected strontium, fluoride, and parathyroid extract. J. Dent. Res., 42: 1208-1216. Yoshida, H.,D. Nagai, M. Kamei and Y. Nakagawa 1968 Irreversible inactivation of (Naf - K+)-dependent ATPase and K+-dependent phosphatase by fluoride. Biochem. Biophys. Acta, 150: 162-168.
PLATE 1 EXPLANATION O F FIGURE
1
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Control secretory ameloblasts. This electron micrograph is from inner enamel. Note the axially oriented granular endoplasmic reticulum ( E ) , large dark and light granules ( G ) , mitochondria ( M ) , vacuoles ( V ) , terminal cell web ( W ) , and fibril (F). Magnification x 5,495.
ION INDUCED DISTURBANCES I N AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
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2
Ameloblasts two hours after injection with fluoride. This montage shows numerous membrane-bound large vacuoles and dark globules. Note the variety of sizes and densities of the spherical bodies. Some endoplasmic reticulum is displaced by these bodies. V, vacuoles; D, dark globules; E, endoplasmic reticulum. x 3,895.
3
Tomes’ process eight hours after injection with fluoride shows accumulation of larger lakes of stippled material. x 3,420.
4
Ameloblasts 24 hours after injection with fluoride. The cells appear normal. Note alterations in size and morphology of Tomes’ process. T, Tomes’ process. X 3,325.
5
Enamel formed 48 hours after fluoride injection. The hypomineralized band is overlaid by normal prism structure. 0, hypomineralized band. Magnification X 3,040. Inset: The hypomineralized band containing a few scattered crystals. x 6,080.
ION INDUCED DISTURBANCES IN AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
6
Ameloblast four hours after injection with cobalt. Note globules and vacuoles are enclosed within membranes. Small dense bodies are being formed in the Golgi zone. S, secretion granules; G, Golgi; V, vacuoles; D, dark globules. x 5,400.
7 Tomes’ process eight hours after injection with cobalt. Stippled material accumulates at tips of the enamel prongs and distal end of the process (arrows). x 5,450. 8
Tomes’ process 24 hours after injection with cobalt. Note lakes of non-crystalline stippled material forming a circumferential outline around the prism (arrows). x 5,100.
9 Enamel structure formed 48 hours after cobalt injection. Normal prism structure is formed over the hypomineralized region. Enamel crystal formation is not observed in the hypomineralized region. X 5,100.
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PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
10
Montage of ameloblasts four hours after injections with strontium. Large globules and vacuoles appear stacked up in the distal half of the cell. G, globules; V, vacuoles; S, stippled material. x 4,590.
11 Ameloblasts from animal injected 48 hours previously with strontium.
Note the severe alterations in the morphology of Tomes’ process and the accumulation of stippled material at the distal ends of the process. Enamel on either side of the first hypomineralized region shows some regions of increased electron density and a lack of prism morphology. T, Tomes’ process; S, stippled material; 0, hypomineralized region. x 4,050.
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PLATE 1
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PLATE 5 EXPLANATION O F FIGURE
12 Higher magnification of enamel from area similar to figure 11. Huge lakes of stippled material accumulate at the intercellular spaces. S , stippled material. x 10,500.
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PLATE 5
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0
w
w
Microradiograph of enamel 48 hours after injection with fluoride. A n outer hypomineralized layer (dark) is formed above an inner hypermineralized layer ( b e tween arrows). On the outermost surface is a layer of normal enamel. x 190.
16 Microradiograph of incisor two weeks after injection with strontium. Note the lack of recovery in the affected area occupying most of the microradiograph and the region of later-forming normal enamel ( n ) on the far right (dentino-enamel junction, dark arrow). x 50.
15 Microradiograph of enamel 48 hours after injection with strontium. Note alternating rows of hyper- ( e r ) and hypomineralized ( 0 ) enamel (arrows). x 190.
14 Microradiograph of enamel response 48 hours after injection with cobalt. A n outer hypermineralized layer (light) is formed above an inner hypomineralized layer (dark). On the outermost surface is a layer of normal enamel. x 190.
13
PLATE 6 EXPLANATION O F FIGURES
ION INDUCED DISTURBANCES IN AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 6
This investigation examined ultrastructurally the entire period of development of alterations in formative ameloblasts and the enamel which they produce following injection with fluoride, strontium, and cobalt ions. Rats injected with these ions were sacrificed at intervals of 1, 2, 4, 8, 16,24 and 48 hours to elucidate the sequence and detail of cytologic and cell product alterations which occur. Undecalcified sections of rat incisor teeth were studied using electron microscopy and microradiography. All three ions initially produced disturbances in cell morphology and enamel formation consisting of dark globules, vacuoles, and pooling of stippled material on the enamel surface. While a period of decreased crystal formation occurred after injection with all three ions, only cobalt responses included a period of apparently complete absence of crystd formation. The hypermineralized layers occurring in the altered enamel are attributed to changes in the rate of enamel matrix formation and duration of its exposure to tissue fluids. Morphologic changes in Tomes' process were observed at the time of formation of abnormal enamel following injection of all three ions. These observations are compared with previous studies of altered enamel formation and analyzed with the goal of learning more about the mechanisms of amelogenesis. ABSTRACT
Schour and Smith, '34, '35) showed that subcutaneous injections of sodium fluoride caused disturbances in enamel formation as layers of hypomineralized and hypermineralized enamel. They observed large darkly stained globules in the ameloblast a few hours following injection. Several light and electron microscopic studies since then have also reported morphological changes in the formative ameloblast induced by fluoride (Bhussry, '59; Kruger, '67, '68, '70; Walton and Eisenmann, '72). These changes consistently include large, darkly stained globules and large vesicles in the distal region of the cell body. Weinmann ('43) reported that injected strontium caused enamel hypoplasia and hypomineralization. The ameloblasts were seen to undergo degenerative changes and detachment from the enamel matrix. Recent electron microprobe studies of the effect of dietary strontium on forming enamel revealed a n increased uptake of strontium and decreased uptake of calcium and phosphate (Johnson and Singer, '67). ANAT.REc., 183: 303-322.
Microradiography has been used to assess mineralization disturbances induced by fluoride, strontium, and cobalt (Allen, '63; Weber and Yaeger, '64; Yaeger, '66; Eisenmann and Yaeger, '69; Kruger, '71). Fluoride produced a double response i n rat enamel consisting of a n inner (first formed ) hypermineralized layer followed by a n outer layer. Cobalt produced first a hypomineralized layer and then a hypermineralized layer, the inverse sequence of fluoride. Strontium produced a hypermineralized enamel followed by a diffuse continuous hypomineralized area. Cell damage has been suggested as being responsible for all developmental mineralization lesions except those caused by amdogenesis imperfecta (Nylen et al., '72). Through electron microscopy, disturbances in rat enamel induced by injections of tetracycline were shown to result from a change in rate of matrix secretion Received Aug. 22, '74. Accepted Apr. 3, '75. 1This study was supported in part by grant PHS DE03312 from the National Institute for Dental Research.
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and disturbances in nucleation. The inner hypermineralized layer was thought to be caused by uninterrupted ion transport to previously nucleated crystals during the period when the hypomineralized layer was formed. Disturbances in crystal growth were attributed to the inability of the injured secretory ameloblast to fully recover and function normally during its maturative phase. Previously cited studies have examined alterations in enamel and changes in morphology of cells following various inducing agents. These studies were limited in that they usually considered these disturbances only after they were completed. Investigators have usually studied either the alterations of cellular morphology or variations in enamel density, but with little correlation of these two aspects. This study examines the ameloblasts and their product during the entire period of apposition of altered enamel, utilizing parenteral dosages of strontium, cobalt, and fluoride known to induce disturbances in enamel formation (Eisenmann and Yaeger, '69). Its purpose is to gain further insight into some of the mechanisms of amelogenesis. MATERIALS AND METHODS
Electron microscopy Male Sprague-Dawley rats weighing 175-250 g were given a single subcutaneous injection of either 5 m g sodium fluoride, 125 mg strontium chloride (Yaeger and Eisenmann, '63), or 100 m g cobalt chloride/100 g body weight (Eisenmann and Yaeger, '69) and sacrificed at intervals of 1, 2, 4, 8, 16, 24 and 48 hours. A minimum of two animals was utilized for each time interval. This time period covers the development of the altered enamel produced by the secretory ameloblasts. Control animals were given identical dosages of sodium chloride and were sacrificed at 1, 2 and 4 hours. Our control animals plus controls done in a previous study (Eisenmann and Yaeger, '69) at longer time intervals confirm that the enamel response is not a result of the injection procedure itself. The rats were anesthetized with ether and fixed by vital perfusion through the aorta with 4% gluteraldehyde in 0.04 M cacodylate buffer
at pH 7.2. The maxillary incisors were reremoved along with the surrounding alveolar bone keeping the enamel organ intact. [ncisors were mounted on a Gillings-Hamco sectioning machine and longitudinal sections of 150-300 were cut through the middle third using normal saline solution as a coolant. These sections were returned to cold gluteraldehyde for one hour, rinsed in cacodylate buffer for one hour, and post-fixed in veronal buffered 1% osmium tetraoxide for one hour. Specimens were then dehydrated in a graded series of alcohols and flat-embedded in araldite for cross-sectioning. Sectioning was one with a Porter-Blum microtome equipped with a diamond knife. The sections were taken from the region where inner enamel had reached a thickness of 20-50 pm. Thick sections (0.5 pm) were examined in the light microscope to assist in achieving the proper location and orientation. Ultra-thin sections were floated on a saturated solution of dibasic calcium phosphate (Boothroyd, '64), collected on parloidin and carbon-coated grids and stained with uranyl magnesium acetate and lead citrate. The specimens were examined with a Hitachi 7-S electron microscope at 50 kv.
Microradiography Forty-eight hour specimens of cobalt, strontium, and fluoride were embedded in methyl methacrylate. Two week specimens of strontium were similarly processed. Longitudinal sections of 80-100 pm in thickness were cut from the middle third of the tooth with a Gillings-Hamco sectioning machine. Microradiographs were obtained on Kodak 649-0 spectroscopic plates using a nickel filter in a Siemens x-ray generator operated at 19 kV. OBSERVATIONS
Electron microscopy The typical morphology of a tall columnar ameloblast equipped with abundant organelles for protein synthesis and secretion were observed i n all control specimens (fig. 1). 1. Fluoride The morphology of the ameloblast maintains a normal appearance one hour after
ION INDUCED DISTURBANCES IN AMELOGENESIS
injection with fluoride. After two hours numerous large vacuoles and dark globules appear to be stacked-up in the distal half of the cell body (fig. 2). These spherical bodies vary in size and density and are often large enough to span nearly the entire width of the cell, displacing profiles of endoplasmic reticulum. The bodies are membrane-bound and do not appear to communicate with each other or with any other organelle. The organelles of the cell body are of a normal appearance and the quantity of secretion granules is similar to that of the control cells. No alterations appear in Tomes’ process or the adjacent enamel crystals a t this stage of the response to fluoride. After four hours, the dark globules and vacuoles increase in number and in some cases extend to the nuclear region. Small pools of stippled material accumulate just outside of the distal end of Tomes’ process. Other than the large bodies, the general appearance of the components of the cell body and Tomes’ process appear unaltered four hours after fluoride. At eight hours, larger lakes of stippled material form a t the distal ends of Tomes’ processes (fig. 3). There is a decrease in enamel formation between the processes. The large bodies seen earlier in the cell body have decreased in number and size. At 16 hours the cells appear to have nearly recovered from the effects of fluoride. The dark globules and vacuoles seen i n the central region of the ameloblasts are similar in size and amount to those seen i n the control cells. No alterations are present in any of the organelles, but the morphology and size of Tomes’ process has been altered. Enamel formation has resumed between the Tomes’ processes and a t the distal end of each process leaving behind large lakes of noncrystallized or sparsely crystallized stippled material. At 24 and 48 hours after the injection the ultrastructure of the ameloblast shows no unique features other than a continued alteration after 24 hours in Tomes’ process (fig. 4 ) . Normal enamel is being laid down over the hypomineralized band. The layer of altered enamel consists of areas containing scattered disoriented crystals in a homogeneous osmiophilic background and spaces devoid of mineral but contain-
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ing a loosely arranged granular material (fig. 5). 2. Ctjbalt The ameloblast maintains a normal appearance two hours after injection with cobalt. Small pools of stippled material lacking crystals accumulate just outside of the distal and mid-proximal regions of Tomes’ process. At four hours after injection, numerous large vacuoles and dark globules varying in size and density appear in the distal third of the cell body (fig. 6). The quantity and size of the spherical bodies are similar to those seen after injection of fluoride. After eight hours the large spherical bodies seem to have decreased in size and number. The morphology and size of Tomes’ process is altered. Lakes of stippled materisl accumulate a t the distal end of Tomes’ process (fig. 7). At 24 and 48 hours after the injection, the ultrastructure of the ameloblast appears normal. Crystal formation occurs after 24 hours over the altered hypomineralized regions leaving behind lakes of non-crystalline stippled material (fig. 8). A resumption of normal enamel formation occurs overlying the hypomineralized region (fig. 9 ) . The thickness of enamel formed over the hypomineralized region is less than the amount formed after the fluoride response (fig. 5).
Strontium One and two hours after injection with strontium, the ultrastructure of the ameloblast reflects no morphological differences from control cells. After four and eight hours, large vacuoles and dark globules accumulate in the distal half of the cell body (fig. 10). Small lakes of stippled material pool at the distal end of Tomes’ process and along the enamel spikes. At eight hours, the enamel front exhibits a disorganization of crystals and a lack of prism morphology. At 16 hours, the spherical bodies decrease in size and amount. Observations of the organelles reveal nothing abnormal. Pools of stippled material accumulate in the intercellular spaces between the processes along with a decrease in the formation of interprismatic enamel in these 3.
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areas, A broad spectrum of alteration is noted in the size and morphology of the Tomes’ process of some cells. The normal prismatic pattern of enamel is not evident in such areas. At 24 hours, large lakes of stippled material accumulate on the enamel surface and more proximally between the Tomes’ processes. Large vacuoles and dense globules again appear in the distal cytoplasm. Tomes’ processes are severely altered in size and morphology and the adjacent enamel contains scattered, disorganized crystals. At 48 hours after injection, numerous large vacuoles and dark globules varying in size and density persist in the distal half of the cells (fig. 11). The distal end shows a reduction of Tomes’ process into thin finger-like structures of varied shapes. Huge lakes of non-crystalline, dense homogeneous material accumulate in the intercellular spaces between ameloblasts on the surface of the enamel (fig. 12). The mineralizing front contains a decreased concentration of crystals which are haphazardly organized (fig. 11). This area represents formation of a second hypomineralized layer. On either side of the first-formed hypomineralized band, there is a lack of prism morphology. The organic matrix observed in the first-formed hypomineralized regions and that which is accumulating at the enamel front consists of a n osmiophilic material containing a few scattered crystals.
Microradiograp hy Following are microradiographic descriptions of the response to each ion at 48 hours after injections in the formative stage of amelogenesis : Fluoride. The response to fluoride consists of two components: a n inner (firstformed) hypermineralized band, and a n outer hypomineralized component covered with a thin layer of newly formed normal enamel (fig. 13). Cobalt. The response to cobalt consists of a n inner hypomineralized region which precedes a n outer hypermineralized layer (fig. 4). On the surface is a layer of newly formed normal enamel. Strontium. The response seen in the microradiograph consists of up to four
components in some regions. Proceeding from the inner (first-formed) layer outward they are : 1. a n inner hypennineralized layer; 2. a hypomineralized layer; 3. a hypermineralized layer; 4. a n outer hypomineralized region, Little, if any, normal enamel is formed external to the outer hypomineralized layer (fig. 15). An additional %week follow-up examination of the strontium response revealed a complete lack of resumption of normal enamel secretion in the affected region with normal enamel formation by ameloblasts developing later (fig. 16). DISCUSSION
Electron microscopic investigation of amelogenesis disturbed by strontium, fluoride, and cobalt has revealed alterations i n both cell morphology and enamel formation. Early changes following injection with all three ions consist of accumulations of dark globules and vacuoles in the cell and pooling of stippled material on the enamel surface. Permanent recovery of enamel formation occurs after cobalt and fluoride, but the brief recovery by the ameloblasts after injection with strontium is followed by another severe response in the cell and enamel after 48 hours and a lack of subsequent recovery in the affected area. Johnson et al. (’68) have demonstrated that strontium incorporated in the bone of rats is subsequently translocated to the incisor teeth during the remodeling process of bone. This process may be responsible for the second wave of disturbance following strontium injection. The orientation of enamel crystals and the resultant formation of a highly ordered prismatic structure has been attributed to the organizing properties of the Tomes’ process cell membrane (Boyde, ’64). Variations in Tomes’ process were observed in ameloblasts at the time of formation of abnormal enamel following injection of all three ions. The severest alteration of Tomes’ process was seen after injection with strontium which also produced the most extreme changes in enamel structure. The least severe disturbance of Tomes’ process was produced by cobalt which induced the mildest defect insofar as prism morphology is concerned. Variations in enamel structure are also seen during nor-
ION INDUCED DISTURBANCES I N AMELOGENESIS
ma1 development when formation has first begun at the dentino-enamel junction and when it is nearly completed producing layers of prismless enamel. At both of these stages of enamel formation Tomes' processes are undergoing marked changes in morphology : either developing as cell processes or being phased out. This information along with the currently observed relationship between Tomes' process morphology and the quality of enamel structure, support an organizational function for Tomes' process. A number of factors must be considered in analyzing mechanisms of interference with enamel formation. Among these are quality of matrix, crystal nucleation and growth, transfer of mineral ions to the matrix and systems acting as regulators of mineralization. Although it has been demonstrated autoradiographically (Kruger, '70) that some variation in enamel matrix occurs following fluoride injection, poorly mineralized areas OE dentin induced by strontium and fluoride were found capable of accumulating mineral in vitro (Eisenmann and Yaeger, '72). This indicates that the organic matrix, if it was altered, was at least not permanently resistant to mineralization. The pools of unmineralized stippled material observed in the present study as well as the regions of sparsely mineralized matrix are present at times when mineral is being deposited in adjacent hypermineralized zones of the strontium and fluoride responses. The resistance to mineralization by specific regions may be due to localized interference with certain of the other factors involved in the mineralization process and known to be susceptible to interference by at least some of the ions injected. For example, enzyme systems essential for proper cell function are inhibited by fluoride (Frajola, '59; Yoshida et al., '68); and cobalt, fluoride and strontium are reported to have various inhibitory effects on crystal nucleation and growth (Bird and Thomas, '63). In addition, it has been suggested by Russell and Fleisch ('70) that fluoride may exert its effect on mineralization indirectly by inhibiting phosphatase and permitting a greater action by pyrophosphate, a known inhibitor of mineralization.
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Recent evidence points toward the active involvement of formative cells in transferring mineral ions from the circulating fluids to various mineralizing tissues (Kashiwa and Mukai, '71; Martin and Matthews, '70; Kuhar, '74). If the ameloblast is a mineral transporting cell, it is conceivable that the injected ions could interfere with this process by enzyme disturbances, membrane alterations, or by calcium and phosphate binding within the cell. Fluoride-induced enamel changes appear to consist of localized interference with mineralization of newly formed matrix with concurrent accumulation of available calcium and phosphate ions in the preresponse enamel. This pattern of formation of hypermineralized enamel has been proposed by previous authors (Weber and Yaeger, '64; Nylen et al., '72). Only isolated areas of matrix in the hypomineralized layer of some specimens appear to be so severely altered morphologically as to prevent mineralization completely. Those areas of matrix which do contain crystals but of a deficient number appear otherwise similar to normal enamel matrix. Thus it is likely that mineral ion flow has continued and a brief localized alteration in mineralization of newly forming matrix has occurred. This may be a result of inhibition of enzymes such as phosphatase and/or interference by fluoride with the processes of crystal nucleation and growth. Complete recovery of the ameloblast is indicated by the substantial thickness of normal enamel deposited over the hypomineralized layer. The response to strontium may be considered as a double fluoride response. Both ameloblastic and enamel alterations g o through two cycles which, as mentioned previously, may reflect a secondary wave of strontium ions as they are released from bone, Strontium eventually leads to a cessation of enamel formation which is evidenced by the lack of post-response enamel observed microradiographically two weeks after injection. The matrix produced shortly after cobalt injection remains almost completely devoid of crystals. It is interesting to note that the appearance of this unmineralized ma-
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trix is no different from that of normal stippled material. Also it is significant that no hypermineralization occurs in the preresponse enamel. These observations lend support for speculation that a temporary interruption in mineral ion flow may occur. However, it seems unlikely that this alone could be responsible for the hypomineralized layer, because when ion flow is resumed it would lead to crystal formation in the older crystal deficient regions as well as the newly formed matrix. As a known inhibitor of nucleation (Bird and Thomas, '63), cobalt may be present in the hypomineralized region and thus prevent its mineralization even after ion flow resumes. The formation of a subsequent hypermineralized layer may well be due to a period of decreased rate of enamel matrix formation as evidenced by the lesser amount of post-response enamel as compared to after fluoride. Ion induced disturbances i n amelogenesis lead to various combinations of cellular and enamel defects which have many aspects in common along with certain major variations. Nylen et al. ('72) have proposed that most disturbances in ameloblast function follow a similar pattern one that is comparable to that observed here for fluoride and strontium. However, i t appears that the response to cobalt is unique and may lend itself to further study of some of the intricate mechanisms by which normal enamel is formed. ACKNOWLEDGMENTS
The authors wish to express their appreciation to Mrs. Elena Baltrusaitis for her technical assistance and Mrs. Izabele Stoncius for her photographic work. LITERATURE CITED Allan, J. H. 1963 Observations o n the development of dental enamel in acute experimental fluorosis. In: Advances in Fluorine Research and Dental Caries Prevention. J. L. Hardwick, J . P. Destin and H. R. Held, eds. The Macmillan Co., New York, pp. 41-51. Bhussry, B. R. 1959 Effects of sodium fluoride on the developing teeth of rats. J. Dent. Res., 38: 653-654. Bird, E. D., and W. C. Thomas, Jr. 1963 Effect of various metals on mineralization in vitro. Proc. SOC.Exp. Biol. Med., 112: 640-643. Boothroyd, B. 1964 The problem of demineral-
ization in thin sections of fully calcified bone. J. Cell Biol., 20: 165-175. Boyde, A. 1964 The Structure and Development of Mammalian Enamel. Ph.D. Thesis, London Hospital Medical College, London. Eisenmann, D. R., and J. A. Yaeger 1969 Alterations in the formation of rat dentin and enamel induced by various ions. Archs. Oral Biol., 1 4 : 1045-1064. 1972 In vitro mineralization of hypomineralized dentin induced by strontium and fluoride. Archs. Oral Biol., 27: 987-999. Frajola, W. J. 1959 Fluoride and enzyme inhibition. In: Fluorine and Dental Health. The Pharmacology and Toxicology of Fluorine. J. C. Muhler and M. K. Hine, eds. Indiana University Press, Bloomington, pp. 68-69. Johnson, A. R., W. D. Armstrong and L. Singer 1968 The incorporation and removal of large amounts of strontium by physiologic mechanisms ni mineralized tissues of the rat. Calc. Tiss. Res., 2: 242-252. Johnson, A. R., and L. Singer 1967 A n electronmicroprobe study of rat incisor teeth with low or high concentrations of strontium. Archs. Oral Biol., 12: 389-399. Kashiwa, H. K., and C. D. Muai 1971 Lipidcalcium-phosphate spherule i n chondrocytes of developing long bones. Clin. Orth., 78: 223-229. Kruger, B. J. 1967 Histologic effects of fluoride and molybdenum on developing dental tissues. Aust. Dent. J., 12: 54-60. 1968 Ultrastructural changes in ameloblasts from fluoride treated rats. Archs. Oral Biol., 13: 969-977. 1970 An autoradiographic assessment of the effect of fluoride on the uptake of H3-proline by ameloblasts in the rat. Archs. Oral Biol., 15: 103-108. 1970 The effect of different levels of fluoride on the ultrastructure of ameloblasts in rat. Archs. Oral Biol., 15: 109-114. 1971 Scanning electron microscopy of sections of fluorosed rat enamel. J. Dent. Res., 50: 1685. Kuhar, K. J. 1974 Fluoride-induced Calcification within the Maturative Ameloblast of the Rat. M. S. Thesis, University of Illinois, Chicago. Martin, J. H., and J. L. Matthews 1970 Mitochondrial granules in chondrocytes, osteoblasts and osteocytes. Clin. Orth., 68: 273-278. Nylen, M., K. Ommell and C. Lofgren 1972 A n electron microscopic study of tetracyclineinduced enamel defects in rat incisor enamel. Scand. J. Dent. Res., 80: 384-409. Russell, R. G., and H. Fleisch 1970 Inorganic pyrophosphate and pyrophosphatases in calcification and calcium homeostasis. Clin. Orth., 69: 101-117. Schour, I., and M. C. Smith 1934 The histologic changes in the enamel and dentin of the rat incisor i n acute and chronic experimental fluorosis. Univ. Ariz. Coll. Agric., Agric. Exp. Sta., Techn. Bull., 52: 67-91. 1935 Mottled teeth: An experimental histologic analysis. J. Am. Dent. Assoc., 22: 796-8 13.
ION INDUCED DISTURBANCES IN AMELOGENESIS Walton, R. E., and D. R. Eisenmann 1974 Ultrastructural examination of various stages of amelogenesis in the rat following parental fluoride administration. Archs. Oral Biol., 19: 171-183. Weber, D. F., and J. A. Yaeger 1964 A microradiographic interpretation of abnormal enamel formation induced by subcutaneous sodium fluoride. J. Dent. Res., 43: 50-56. Weinmann, J. P. 1943 Recovery of ameloblasts. J. Amer. Dent. Assoc., 30: 874-888. Yaeger, J. A. 1966 The effects of high fluoride
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diets on developing enamel and dentin in the incisors of rats. Am. J. Anat., 18: 665-683. Yaeger, J. A,, and D. R. Eisenmann 1963 Response in rat incisor dentin to injected strontium, fluoride, and parathyroid extract. J. Dent. Res., 42: 1208-1216. Yoshida, H.,D. Nagai, M. Kamei and Y. Nakagawa 1968 Irreversible inactivation of (Naf - K+)-dependent ATPase and K+-dependent phosphatase by fluoride. Biochem. Biophys. Acta, 150: 162-168.
PLATE 1 EXPLANATION O F FIGURE
1
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Control secretory ameloblasts. This electron micrograph is from inner enamel. Note the axially oriented granular endoplasmic reticulum ( E ) , large dark and light granules ( G ) , mitochondria ( M ) , vacuoles ( V ) , terminal cell web ( W ) , and fibril (F). Magnification x 5,495.
ION INDUCED DISTURBANCES I N AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
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2
Ameloblasts two hours after injection with fluoride. This montage shows numerous membrane-bound large vacuoles and dark globules. Note the variety of sizes and densities of the spherical bodies. Some endoplasmic reticulum is displaced by these bodies. V, vacuoles; D, dark globules; E, endoplasmic reticulum. x 3,895.
3
Tomes’ process eight hours after injection with fluoride shows accumulation of larger lakes of stippled material. x 3,420.
4
Ameloblasts 24 hours after injection with fluoride. The cells appear normal. Note alterations in size and morphology of Tomes’ process. T, Tomes’ process. X 3,325.
5
Enamel formed 48 hours after fluoride injection. The hypomineralized band is overlaid by normal prism structure. 0, hypomineralized band. Magnification X 3,040. Inset: The hypomineralized band containing a few scattered crystals. x 6,080.
ION INDUCED DISTURBANCES IN AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 2
PLATE 3 EXPLANATION OF FIGURES
6
Ameloblast four hours after injection with cobalt. Note globules and vacuoles are enclosed within membranes. Small dense bodies are being formed in the Golgi zone. S, secretion granules; G, Golgi; V, vacuoles; D, dark globules. x 5,400.
7 Tomes’ process eight hours after injection with cobalt. Stippled material accumulates at tips of the enamel prongs and distal end of the process (arrows). x 5,450. 8
Tomes’ process 24 hours after injection with cobalt. Note lakes of non-crystalline stippled material forming a circumferential outline around the prism (arrows). x 5,100.
9 Enamel structure formed 48 hours after cobalt injection. Normal prism structure is formed over the hypomineralized region. Enamel crystal formation is not observed in the hypomineralized region. X 5,100.
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ION INDUCED DISTURBANCES IN AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
10
Montage of ameloblasts four hours after injections with strontium. Large globules and vacuoles appear stacked up in the distal half of the cell. G, globules; V, vacuoles; S, stippled material. x 4,590.
11 Ameloblasts from animal injected 48 hours previously with strontium.
Note the severe alterations in the morphology of Tomes’ process and the accumulation of stippled material at the distal ends of the process. Enamel on either side of the first hypomineralized region shows some regions of increased electron density and a lack of prism morphology. T, Tomes’ process; S, stippled material; 0, hypomineralized region. x 4,050.
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ION INDUCED DISTURBANCES IN AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 1
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PLATE 5 EXPLANATION O F FIGURE
12 Higher magnification of enamel from area similar to figure 11. Huge lakes of stippled material accumulate at the intercellular spaces. S , stippled material. x 10,500.
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PLATE 5
319
0
w
w
Microradiograph of enamel 48 hours after injection with fluoride. A n outer hypomineralized layer (dark) is formed above an inner hypermineralized layer ( b e tween arrows). On the outermost surface is a layer of normal enamel. x 190.
16 Microradiograph of incisor two weeks after injection with strontium. Note the lack of recovery in the affected area occupying most of the microradiograph and the region of later-forming normal enamel ( n ) on the far right (dentino-enamel junction, dark arrow). x 50.
15 Microradiograph of enamel 48 hours after injection with strontium. Note alternating rows of hyper- ( e r ) and hypomineralized ( 0 ) enamel (arrows). x 190.
14 Microradiograph of enamel response 48 hours after injection with cobalt. A n outer hypermineralized layer (light) is formed above an inner hypomineralized layer (dark). On the outermost surface is a layer of normal enamel. x 190.
13
PLATE 6 EXPLANATION O F FIGURES
ION INDUCED DISTURBANCES IN AMELOGENESIS Abraham Neiman and Dale R. Eisenmann
PLATE 6
