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Ultrastructural examination of dentine formation in rat incisors following multiple fluoride injections
Archs
ord
Bid.
Vol.
20. pp
4X5
lo 488.
Pergamon Press 1975. Prmted m Great Brew
ULTRASTRUCTURAL EXAMINATION OF DENTINE FORMATION IN RAT INCISORS FOLLOWING MULTIPLE FLUORIDE INJECTIONS R. E. Department
of Histology.
College
WALTON*
of Dentistry. Illinois
and D. R.
EISEKMANN
University of Illinois at the Medical 60680. U.S.A.
Center
Chicago,
Summary--Rats were divided into two experimental and one control groups. In the first experimental group, two doses were injected each day at 6 hr intervals. The high dosages of sodium fluoride .were administered daily for 5 days. These fluorotic animals were killed as the fluoride was exerting an effect on mineralization. In the other experimental group, the rats were allowed a recovery period by killing the rats 3 days after the last fluoride injections. Control animals received equivalent dosages of sodium chloride and were killed on the 5th day. Each d;iy’s injections induced a consistent paired response in the dentine in both experimental groups-a zone of hypermineralization followed by a zone of hypomineralization. The hype:rmineralized component was consistently visible only on microradiographs as bands of increased density, while the hypomineralized component was evident only on electron micrographs. The fine structure of the hypomineralized zone consisted of patchy areas with the appearance of predentine devoid of crystals and interspersed with calcospherites of mineralized dentine. These globules acquired more mineral with time to fill most areas, leaving unmineralized patches adjacent to odontoblast processes.. In the recovery animals, a few sma.11 regions of unmineralization persisted and a layer of normal dentine formed over the fluoride response zones. Neither fluorotic nor recovery odontoblasts manifested visible ultrastructural alterations.
INTRODUCTION
It has been repea.:edly demonstrated that dentine formation is disturbed following injections of large doses of sodium fluoride. This phenomenon has been examined in rat incisors by administering the sodium fluoride in a single subcutaneous dose (Yaeger, 1963; Yaeger and Eisenmann, 1963; Eisenmann and Yaeger, 1969, 1972) or by repeated intraperitoneal injections for several days (&hour and Smith, 1934; Kruger, 1967). Microradiography of thin ground sections of dentine has consistently revealed a response in the dentine which forms at the time of injection (Yaeger and Eisenmann, 1963; Yaeger, Henrichsen and Cohen. 1964; Osmanski and Yaeger, 1965; Eisenmann and Yaeger, 1969. 1972). The response is paired; an external layer of relative hypermineralization and an adjacent, internal (pulpward) layer of relative hypomineralization. The severity of the response tends to increase in proportion to the dosage of sodium fluoride (Yaeger and Eisenmann, 1963). A time study, utilizing tetracycline as a marker, has demonstrated that both components of the response develop concurrently (Yaeger, et al., 1964). Neither layer is homogeneous; the hypomineralized line exhibits differences ‘in radiolucency in various locations in the cross-section and generally is wider than the hypermineralized component. * Present addrer,s: Departments of Endodontics and Oral Biology. School of Dentistry, Medical College of Georgia. Augusta, Georgia 30902, U.S.A. 4x5
Ultrastructural studies have demonstrated that the hypomineralized regions which develop after a single injection of NaF are less electron-dense than adjacent dentine. When these sections were decalcified, collagen could be identified throughout the specimen (Yaeger. 1963). A more recent investigation (Eisenmann and Yaeger. 1972) of undemineralized fluorotic dentine has disclosed that the hypomineralized regions contain abundant, normal-appearing collagen and isolated accumulations of mineral. There is no published information regarding the effects of injected fluoride on the fine structure of the odontoblast although preliminary work in our laboratory suggested that these cells exhibited no alterations as the result of a single large subcutaneous dose of fluoride. Microradiography has shown that normal dentine formation resp,mes following the disturbances in mineralization induced by a single subcutaneous injection of fluoride. showing that the effects of the ion are temporary (Osmanski and Yaeger. 1964; Yaeger et ul., 1964; Eisenmann and Yaeger. 1969). Also, the hypomineralized zone becomes less distinct as it accumulates mineral while being buried under layers of constantly forming dentine. The hypermineralized layer becomes increasingly obscured as the adjacent normal dentine matures (Yaeger. 1966). There are no published reports of the ultrastructure of odontoblasts or dentine which forms during the recovery period following cessation of fluoride administration. The purpose of the present study was to examine odontoblasts and dentine formed during and after
R.
4X6
multiple sodium there is forming
E. Walton and D. R. Eisenmann
intraperitoneal injections of large doses of fluoride in rats. This may indicate whether a visible accumulative effect of fluoride on dentine and associated odontoblasts.
MATERIALS
AND METHODS
The specific details of the methods and preparation of experimental materials were outlined in a previous publication (Walton and Eisenmann. 1974). Specimens of dentine and odontoblasts were recovered from these same rats. They were grouped as follows: I. Flirorutic a,~bnu/s. Twelve rats were given two intraperitoneal injections daily. 6 hr apart, of 2.5 per cent sodium fluoride, 17 mg/kg- ’ per injection. for a total of 4 days. On the 5th day, the animal was killed 3 hr after a final injection. 2. Rccorcr~~ ~~~~id.s. Ten rats received the same regimen as the fluorotic animals. The injections were discontinued after 4 days and the animals were allowed 3 days to recover before they were killed. 3. Corrtrol uninruls. These were administered the above dosages of sodium chloride intraperitoneally for 4 days and were killed on the 5th day. The rats were fixed by arterial perfusion with 4 per cent glutaraldehyde in 0.5 M cdcodylate buffer. The incisors were dissected from the maxilla and cross-sections (100 pm) of the apical one-third of the incisor to include dentine and pulp were cut on a Gillings-Hamco thin sectioning machine. Specimens for electron microscopy were post-fixed in 1 per cent osmium tetroxide and flat-embedded in Araldite. Undecalcified thin sections were cut with a diamond knife. stained with uranyl magnesium acetate and lead citrate and examined with a Hitachi 7s electron microscope. Ground cross-sections of dentine were microradiographed using Kodak Spectroscopic 649-O plates in a Siemens X-ray generator.
OBSERVATIONS
Consistent, predictable responses were elicited in the dentine of experimental rats by the injections of sodium fluoride. Each response zone consisted of the alterations that coincided with 1 day’s administration, i.e. two daily injections. 6 hr apart were responsible for one response zone. Since the fluorotic animals received the double dose for 4 days and a single dose prior to death on the 5th day, four complete and one developing response zone appeared in the dentine. Recovery animals received the double dose for 4 days; four response zones were elicited. MicroradiogiuphJ
The nature of the alterations in mineral density was clearly demonstrated on microradiographs of crosssections of the incisors (Figs. I and 2). Concentric bands of radiopacity were seen-one band of hypermineralization coincided with each day of injection. The bands of hypermineralization had intervening zones of less density. usually similar in opacity to normal dentine. Occasionally these intervening zones were radiolucent relative to normal dentine, indicating hypomineralization.
The response zones were apparent in both the Ruorotic and recovery dentine. The zones in each experimental group were similar-a band of hypomineralization paired with a band of less mineralization. The fluorotic dentine (Fig. I) contained bands adjacent to its inner border; the innermost hypermineralized band was forming when the rat was killed. The recovery incisor exhibited a rrsponsc zone (Fig. 2) formed during the 4 days of fluoride injections: the normal-appearing region formed during the recovery period.
,4. Drritinr. Examination of the tine structure of newly-formed dentine in the response areas also revealed alterations but with surprising variations from the microradiographs. The fluorotic dentine (Figs. 3 and 4) throughout the thickness of the response zone, lacked the uniformity and density of the underlying pre-fluoride dentine. It consisted of an erratic pattern of mineralization; profiles of collagen were present, but only isolated areas acquired mineral, forming globules of crystals on a background of predentine. These crystalline globules formed predominantly in the intertubular areas and accumulated their crystals concentrically, gradually increasing in dimension in the deeper. or earlier-formed dentine. Occasionally. the mineralization pattern demonstrated a linearity parallel to the mineralization front (Fig. 3). In other areas. the globules were dense as mineral accumulated and obscured the underlying collagen. The regions devoid of crystals usually occurred in proximity to the odontoblast process in the peritubular region. Hypermineralized regions could not be identified in electron micrographs at either low or high magnification. The response zone in recovery dentine. which represented 4 days of fluoride injections. persisted deeply in the dentine (Fig. 5). The response zone was aligned parallel to the mineralizing front and consisted of bands of patches devoid of mineral. One linear band of patches formed during I day of dentine apposition. Interestingly. individual patches persisted in the vicinity of an odontoblast process and tended to be aligned a short distance along its length. As in the fluorotic dentine. no bands of hypermineralization were evident, Between the last formed hypomineralized band and the mineralizing front was a wide zone of dentine of normal appearance and of uniform density. This dentine was laid down during the 3 days following termination of Huoride administration, The mineralizing front appeared to be normal. With time. the unmineralized patches decreased in number and size; the deepest. or first-formed layers. demonstrated fewer patches. The remaining patches resembled islands of predentine surrounded by dentine (Fig. 6). Their chief component was collagen. which had the arrangement and density of predentine (Fig. 7). At the periphery of the patches individual crystals would usually overlay collagen fibres. B. Odor~tohlasts. There were no apparent differences in the fine structure of control, fluorotic and recovery odontoblasts. At the level of resolution of the electron microscope. the cells seemed to be morphologically unaffected by fluoride. Predentine also appeared similar in the three groups.
Examination of dentine formation in rat DISCUSSION Method of jluoriae
administration
As discovered in our laboratory, a single subcutaneous injection of a high dose of sodium fluoride caused a local !severe ulceration. Several of these lesions would seriously impair the health of the animal; therefore, the interperitoneal route was selected for multiple injections. The ion was administered twice daily at a 6 hr interval to prolong its effects. Microradiography Earlier investigations (Yaeger, 1963; Yaeger and Eisenmann. 1963, Osmanski and Yaeger. 1964; Eisenmann and Yaeger, 1972) demonstrated that a single subcutaneous injection of sodium fluoride consistently induces a double responsean external band of hypermineralization and an adjacent. internal band of hypomineralization. On microradiographs, we found the respon:se in mineralization patterns following multiple injections to be similar to the single injection in respect of the formation of a band of hypermineralization corresponding to 1 day’s injection (Figs. 1 and 2). The adjacent band of hypomineralization was seldom visible. These bands of hypomineralization were known to be present, as evidenced by examining the ul trastructure (Figs. 3 and 4). Perhaps the discrepancy !n the previous investigations using a single dose and the present investigation using multiple doses relates to the different modes of administration, i.e. fluoride absorption, activity and clearance may be more rapid following intraperitoneal than following subcutaneous injection. The normal density of the “recovery” dentine indicates that the efI?cts of fluoride were temporary and the cells were callable of resumption of normal dentine formation (Fig. 5). Electron microscopy Interestingly. in developing fluorotic dentine, electron micrographs (Figs. 3 and 4) presented a contradictory appearance to that of microradiographs. Hypermineralized (relative to normal dentine) areas were indiscernable, whereas hypomineralization was striking. In fact. the patches devoid of mineral occupied most of the area in the response zone and globules of crystals occupied proportionately less area in most sections. However, these globules must be relatively dense because, microradiographically. bands of hyperm.neralization occurred through these regions. The composite effect of a thick section (the equivalent of thousands of ultra-thin sections) used for microradiography is obviously a significant factor. Evidently enough dense dentine globules formed within the altered regions to produce layers of high relative density. Yaeger (1963) examined the fine structure of hypermineralized and hypomineralized zones and observed crystals in both components; the hypermineralized contained more ‘norganic crystals per unit than did normal or hypomineralized dentine. which had fewer, but larger crystals. These observations contrast with a later investigation (Eisenmann and Yaeger. 1972) and with the present study, in which the hypomineralized bands had Ioatches which contained few, if any, crystals. In addition, neither areas of hypermineraliza-
4x7
tion nor variations in crystal size were observed ultrastructurally. The ultrastructure of the “recovery” dentine (Fig. 5) confirmed the impression of microradiographs (Fig. 2) that normal-appearing dentine forms after fluoride injections are terminated. A similar increase in radiodensity was observed by other investigators (Osmanski and Yaeger, 1965; Yaeger, 1966). As observed on electron micrographs, globules of crystals increased in size with time. Comparing the relative mineral patterns in Figs. 3 and 4 with Figs. 5 and 6. it is obvious that the size of the unmineralized patches steadily decreased. even as they became buried. In confirmation, Eisenmann and Yaeger (1972) recently reported that soon after these hypomineralized regions form, they arc capable of substantial mineral uptake. Possible
mechanisms of fluoride
disturbance
A possibility is alteration of the organic matrix by the presence of fluoride in the hypomineralized areas. or the secretion of an altered organic matrix by affected odontoblasts. Matrix changes-following single injections of fluoride-were shown with autoradiography (Yaeger. 1963 Gady and Yaeger. 1965). However, a recent investigation (Eisenmann and Yaeger. 1972) demonstrated that the hypomineralized matrix contains abundant, apparently normal collagen fibres that are immediately capable of substantial mineral uptake in vitro. The present study also indicates that the matrix per se is not permanently altered by the fluoride ion because hypomineralized regions contained collagen fibrils which steadily accumulated mineral (Figs. 6 and 7). These observations do not, however, rule out the possibility of subtle chemical changes in the matrix, rendering it temporarily unmineralizable. These changes would be reversible, allowing gradual mineral uptake. A possible factor could be the disturbance of phosphoprotein synthesis by the odontoblasts and/ or transport of phosphoproteins to the mineralizing front. Phosphoprotein appears to be synthesized by the odontoblast and has been reported (Weinstock and Leblond, 1973; Butler. Finch and Desteno. 1973) to constitute approximately 10 per cent of rat incisor dentine. This substance may serve as nucleation centres for initial deposition of crystals (Veis and Perry. 1967; Weinstock and Leblond. 1973). Relative to matrix alterations, some interesting comparisons can be made of the effects of fluoride on amelogenesis and dentinogenesis. Enamel and associated cells were taken from the same animals, as reported in a previous study (Walton and Eisenmann. 1974). Although the formative ameloblast manifests some structural alterations while under the influence of fluoride, it continues to secrete enamel matrix. Portions of the matrix are altered and do not acquire the normal number or demonstrate the customary uniform orientation of crystals. An area of fluoride response in enamel is visible in Fig. 6. In contrast, the odontoblast demonstrates no structural alterations. nor does it secrete a matrix which fails to mineralize with time. Another interesting possibility is the inhibition by fluoride of the mechanism which removes inorganic pyrophosphate from the matrix. This substance is present in high concentrations in dentine matrix
Examination
of dentin- frJrmation in rat
A.O.B.
f.p. 488
R. E. Walton and D. R. Eisenmann
Plate 2. Fig. 5. Electron micrograph of recovery dentine. This region corresponds to response zone seen in Fig. 2. The lastformed fluoride-response band is evident. An increasing uptake of mineral has eliminated most of the hypomineralized zone (0); the diminished unmineralized patches have persisted adjacent to the odontoblast processes (OP). Hypermineralization is not evident. Normal-appearing dentine (RD) has formed in the 3 days after cessation of fluoride injections. The mineralizing front (MF) appears normal. Ptedentine (PR). x2600 Fig. 6. A specimen taken from a recovery animal. An early-formed fluoride response zone lies near the dentineenamel junction (DEJ). The hypomineralized component persists as scattered patches largely devoid of crystals. Zones of hypomineralization (0) are evident in the overlying enamel (E). The bands in the enamel formed in response to fluoride administration concurrent to the formation of bands of hypomineralization in dentine (D). x 4000 Fig. 7. Higher magnification of the large unminera!ized patch in the dentine in Fig. 6. It consists primarily of collagen fibres with a similar arrangement and density to predentine. At the periphery, crystals overlie collagen profile (arrows). This patch also abuts an odontoblast process (OP); a portion is cut in tangential section. x 24,600
488
R. E. Walton
and D. R. Eisenmann
(Bisaz. Russell and Fleisch. 1968). It has been speculated (Fleisch and Russell, 1970; Woltgens, Bonting. and Bijvoet, 1971) that inorganic pyrophosphate must be locally destroyed by enzymes, such as pyrophosphatases or alkaline phosphatases, before dental mineralization can occur. The presence of fluoride. a potent inhibitor of both enzyme systems (Graham. Russell and Fleisch, 1970). could temporarily prevent the enzymes from removing pyrophosphate and thereby inhibit calcification. As demonstrated in recovery specimens (Figs. 2 and 5). the levels of fluoride were not sufficiently toxic to damage or permanently alter odontoblast function. therefore altered patterns of the hypomineralization were temporary and resumed when fluoride was cleared. Ackrlo~~lrdgrmolrs-The contributions of time. patience and technical assistance by the staff of the Department of Histology. and in particular Mrs. Elena Baltrusaitis, is greatly appreciated. The research was supported by NIDR Grant DE 03312.
REFERENCES
Bisaz S., Russell R. G. G. and Fleisch H. 1968. Isolation of inorganic pyrophosphate from bovine and human teeth. Arch ord Biol. 13, 6X3-696. Butler W. T.. Finch J. E. and Desteno C. V. 1972. Chemical character of proteins in rat incisors. Biochrm. Biophys. Artn 251, 167-171. Eisenmann D. R. and Yaeger J. A. 1969. Alterations in the formation of rat dentine and enamel induced by various ions. Arc/is orul Bid. 14, 104551064. Eisenmann D. R. and Yaeger J. A. 1972. In vitro mineralization of hypomineralized dentine induced by strontium and fluoride in the rat. .4rchs orul Biol. 17, 9X7-999. Fleisch H. and Russell R. G. G. 1970. Pyrophosphate and polyphosphate. In: I~~tcrruztiorul Encycloprdiu of Pharmacoloyy ud Thcrqwutics pp. 6lIIOO. Pergamon Press. Oxford.
Grady J. E. and Yaeger J. A. 1965. Polarizing microscopy of abnormal dentine produced by injections of strontium or fluoride. Arch oral Biol. 10. 175- 178. Graham R.. Russell G. and Fleisch H. 1970. Inorganic pyrophosphate and pyrophosphatases in calcification. and calcium homeostasis. C/in. Orthop. 69, 101-l 17. Kruger B. J. 1967. Histologic effects’of fluoride and molybdenum on developing dental tissues. Amt. dent J. 12. 5460. Osmanski C. P. and Yaeger J. A. 1964. Microradiography of rat incisor dentine during the development of the responsc to injected fuoride. hat. Rrc. 148. 467-483. Osmanski C. P. and Yaegcr J. A. 1965. The effect of parenteral Ruoride on rat incisor dentine as studied with microradiography. J. Orul Tlier. 1, 650. Schour 1. and Smith M. C. 1934. The histologic changes in the enamel and dentine of the rat incisor in acute and chronic experimental fluorosis. Liaitl. of Ark. Coil. .4yric., Ayric. Ezp. Stu.. Tech Bull. 52, 67-9 1. Veis A. and Perry A. 1967. The phosphoprotein of the dentine matrix. Biochmistr?: 6, 7409-2415. Walton R. E. and Eisenmann D. R. 1974. Ultrastructural examination of various stages of amelogenesis following parenteral fuoride administration. Arch oral Biol. 19. 171&182. Weinstock M. and Leblond C. P. 1973. Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentine of the rat incisor. /. Cell Biol. 56, 838845. Woltgens J. H., Bonting S. L.. Bijvoet 0. L. 1971. Relationship between alkaline phosphatase. inorganic pyrophosphatdse and L-ascorbic acid in calcifying hamster molars. C/in. Orthop. 78, 296301. Yaeger J. A. 1963. Microscopy of the response of rodent dentin to injected Houride. Anut. Rec. 145, 139-147. Yaeger J. A. 1966. Recovery of rat incisor dentine from abnormaI mineralization produced by strontium and huoride. tlnut. Rec. 154, 661-674. Yaeger J. A. and Eisenmann D. R. 1963. Response in rat incisor dentine to injected strontium, fluoride and parathyroid extract. J. dmt. Rex 42, 120881216. Yaeger J. A., Hinrichsen C. F. L. and Cohen M. J. 1964. Development of the response in rat incisor dentine to injected strontium and fluoride. Am. J. Amt. 114. 255271.
Fig. I. Microradiograph of a cross-section of an incisor from a fluorotic rat which received daily injections of sodium fluoride for 5 days and was killed on the 5th day. Four fully developed concentric bands of hypercalcification encompass the pulp space (P). (Insert) A narrow inner band of hypercalcification (arrow) is developing at the mineralizing front. Zones of hypomineralization are not evident in the area between the bands of increased density. Labial cap of enamel (E). Dentine formed prior to fluoride injections (D). Differences in radiodensity between mesial (M) and distal (Ds) components relate to differences in the gross thicknesses of the ground section. x 80 Fig. 2. Microradiograph of an incisor from a recovery animal. The four concentric bands of hypermineralization persist but are buried under a layer of dentine of normal-appearing density (insert). This recovery dentine (RD) adjacent to the pulp space was formed during the 3 days following the last administration of fluoride. x 80 Fig. 3. Electron micrograph of the mineralizing front of fluorotic dentine. This region corresponds to the response zone of Fig. 1. Pre-injection dentine (D) exhibits normal density. The region which was mineralizing at the time of fluoride administration demonstrates an erratic pattern of mineral uptake. Globules of dentine form but fail to coalesce to fill in and obscure the underlying predentine. Compare to the normal mineralizing front as demonstrated in Fig. 5. The linear pattern of the unmineralized regions is noticeable but not prominent, neither is the hypermineralized component (obvious on microradiographs) visible. Predentine (PR). Odontoblast process (OP). x 2400 Fig. 4. This region also corresponds to the response zone of Fig. 1. Occasionally areas are encountered where the unmineralized patches are scattered at random, demonstrating no linearity. x 2400
ord
Bid.
Vol.
20. pp
4X5
lo 488.
Pergamon Press 1975. Prmted m Great Brew
ULTRASTRUCTURAL EXAMINATION OF DENTINE FORMATION IN RAT INCISORS FOLLOWING MULTIPLE FLUORIDE INJECTIONS R. E. Department
of Histology.
College
WALTON*
of Dentistry. Illinois
and D. R.
EISEKMANN
University of Illinois at the Medical 60680. U.S.A.
Center
Chicago,
Summary--Rats were divided into two experimental and one control groups. In the first experimental group, two doses were injected each day at 6 hr intervals. The high dosages of sodium fluoride .were administered daily for 5 days. These fluorotic animals were killed as the fluoride was exerting an effect on mineralization. In the other experimental group, the rats were allowed a recovery period by killing the rats 3 days after the last fluoride injections. Control animals received equivalent dosages of sodium chloride and were killed on the 5th day. Each d;iy’s injections induced a consistent paired response in the dentine in both experimental groups-a zone of hypermineralization followed by a zone of hypomineralization. The hype:rmineralized component was consistently visible only on microradiographs as bands of increased density, while the hypomineralized component was evident only on electron micrographs. The fine structure of the hypomineralized zone consisted of patchy areas with the appearance of predentine devoid of crystals and interspersed with calcospherites of mineralized dentine. These globules acquired more mineral with time to fill most areas, leaving unmineralized patches adjacent to odontoblast processes.. In the recovery animals, a few sma.11 regions of unmineralization persisted and a layer of normal dentine formed over the fluoride response zones. Neither fluorotic nor recovery odontoblasts manifested visible ultrastructural alterations.
INTRODUCTION
It has been repea.:edly demonstrated that dentine formation is disturbed following injections of large doses of sodium fluoride. This phenomenon has been examined in rat incisors by administering the sodium fluoride in a single subcutaneous dose (Yaeger, 1963; Yaeger and Eisenmann, 1963; Eisenmann and Yaeger, 1969, 1972) or by repeated intraperitoneal injections for several days (&hour and Smith, 1934; Kruger, 1967). Microradiography of thin ground sections of dentine has consistently revealed a response in the dentine which forms at the time of injection (Yaeger and Eisenmann, 1963; Yaeger, Henrichsen and Cohen. 1964; Osmanski and Yaeger, 1965; Eisenmann and Yaeger, 1969. 1972). The response is paired; an external layer of relative hypermineralization and an adjacent, internal (pulpward) layer of relative hypomineralization. The severity of the response tends to increase in proportion to the dosage of sodium fluoride (Yaeger and Eisenmann, 1963). A time study, utilizing tetracycline as a marker, has demonstrated that both components of the response develop concurrently (Yaeger, et al., 1964). Neither layer is homogeneous; the hypomineralized line exhibits differences ‘in radiolucency in various locations in the cross-section and generally is wider than the hypermineralized component. * Present addrer,s: Departments of Endodontics and Oral Biology. School of Dentistry, Medical College of Georgia. Augusta, Georgia 30902, U.S.A. 4x5
Ultrastructural studies have demonstrated that the hypomineralized regions which develop after a single injection of NaF are less electron-dense than adjacent dentine. When these sections were decalcified, collagen could be identified throughout the specimen (Yaeger. 1963). A more recent investigation (Eisenmann and Yaeger. 1972) of undemineralized fluorotic dentine has disclosed that the hypomineralized regions contain abundant, normal-appearing collagen and isolated accumulations of mineral. There is no published information regarding the effects of injected fluoride on the fine structure of the odontoblast although preliminary work in our laboratory suggested that these cells exhibited no alterations as the result of a single large subcutaneous dose of fluoride. Microradiography has shown that normal dentine formation resp,mes following the disturbances in mineralization induced by a single subcutaneous injection of fluoride. showing that the effects of the ion are temporary (Osmanski and Yaeger. 1964; Yaeger et ul., 1964; Eisenmann and Yaeger. 1969). Also, the hypomineralized zone becomes less distinct as it accumulates mineral while being buried under layers of constantly forming dentine. The hypermineralized layer becomes increasingly obscured as the adjacent normal dentine matures (Yaeger. 1966). There are no published reports of the ultrastructure of odontoblasts or dentine which forms during the recovery period following cessation of fluoride administration. The purpose of the present study was to examine odontoblasts and dentine formed during and after
R.
4X6
multiple sodium there is forming
E. Walton and D. R. Eisenmann
intraperitoneal injections of large doses of fluoride in rats. This may indicate whether a visible accumulative effect of fluoride on dentine and associated odontoblasts.
MATERIALS
AND METHODS
The specific details of the methods and preparation of experimental materials were outlined in a previous publication (Walton and Eisenmann. 1974). Specimens of dentine and odontoblasts were recovered from these same rats. They were grouped as follows: I. Flirorutic a,~bnu/s. Twelve rats were given two intraperitoneal injections daily. 6 hr apart, of 2.5 per cent sodium fluoride, 17 mg/kg- ’ per injection. for a total of 4 days. On the 5th day, the animal was killed 3 hr after a final injection. 2. Rccorcr~~ ~~~~id.s. Ten rats received the same regimen as the fluorotic animals. The injections were discontinued after 4 days and the animals were allowed 3 days to recover before they were killed. 3. Corrtrol uninruls. These were administered the above dosages of sodium chloride intraperitoneally for 4 days and were killed on the 5th day. The rats were fixed by arterial perfusion with 4 per cent glutaraldehyde in 0.5 M cdcodylate buffer. The incisors were dissected from the maxilla and cross-sections (100 pm) of the apical one-third of the incisor to include dentine and pulp were cut on a Gillings-Hamco thin sectioning machine. Specimens for electron microscopy were post-fixed in 1 per cent osmium tetroxide and flat-embedded in Araldite. Undecalcified thin sections were cut with a diamond knife. stained with uranyl magnesium acetate and lead citrate and examined with a Hitachi 7s electron microscope. Ground cross-sections of dentine were microradiographed using Kodak Spectroscopic 649-O plates in a Siemens X-ray generator.
OBSERVATIONS
Consistent, predictable responses were elicited in the dentine of experimental rats by the injections of sodium fluoride. Each response zone consisted of the alterations that coincided with 1 day’s administration, i.e. two daily injections. 6 hr apart were responsible for one response zone. Since the fluorotic animals received the double dose for 4 days and a single dose prior to death on the 5th day, four complete and one developing response zone appeared in the dentine. Recovery animals received the double dose for 4 days; four response zones were elicited. MicroradiogiuphJ
The nature of the alterations in mineral density was clearly demonstrated on microradiographs of crosssections of the incisors (Figs. I and 2). Concentric bands of radiopacity were seen-one band of hypermineralization coincided with each day of injection. The bands of hypermineralization had intervening zones of less density. usually similar in opacity to normal dentine. Occasionally these intervening zones were radiolucent relative to normal dentine, indicating hypomineralization.
The response zones were apparent in both the Ruorotic and recovery dentine. The zones in each experimental group were similar-a band of hypomineralization paired with a band of less mineralization. The fluorotic dentine (Fig. I) contained bands adjacent to its inner border; the innermost hypermineralized band was forming when the rat was killed. The recovery incisor exhibited a rrsponsc zone (Fig. 2) formed during the 4 days of fluoride injections: the normal-appearing region formed during the recovery period.
,4. Drritinr. Examination of the tine structure of newly-formed dentine in the response areas also revealed alterations but with surprising variations from the microradiographs. The fluorotic dentine (Figs. 3 and 4) throughout the thickness of the response zone, lacked the uniformity and density of the underlying pre-fluoride dentine. It consisted of an erratic pattern of mineralization; profiles of collagen were present, but only isolated areas acquired mineral, forming globules of crystals on a background of predentine. These crystalline globules formed predominantly in the intertubular areas and accumulated their crystals concentrically, gradually increasing in dimension in the deeper. or earlier-formed dentine. Occasionally. the mineralization pattern demonstrated a linearity parallel to the mineralization front (Fig. 3). In other areas. the globules were dense as mineral accumulated and obscured the underlying collagen. The regions devoid of crystals usually occurred in proximity to the odontoblast process in the peritubular region. Hypermineralized regions could not be identified in electron micrographs at either low or high magnification. The response zone in recovery dentine. which represented 4 days of fluoride injections. persisted deeply in the dentine (Fig. 5). The response zone was aligned parallel to the mineralizing front and consisted of bands of patches devoid of mineral. One linear band of patches formed during I day of dentine apposition. Interestingly. individual patches persisted in the vicinity of an odontoblast process and tended to be aligned a short distance along its length. As in the fluorotic dentine. no bands of hypermineralization were evident, Between the last formed hypomineralized band and the mineralizing front was a wide zone of dentine of normal appearance and of uniform density. This dentine was laid down during the 3 days following termination of Huoride administration, The mineralizing front appeared to be normal. With time. the unmineralized patches decreased in number and size; the deepest. or first-formed layers. demonstrated fewer patches. The remaining patches resembled islands of predentine surrounded by dentine (Fig. 6). Their chief component was collagen. which had the arrangement and density of predentine (Fig. 7). At the periphery of the patches individual crystals would usually overlay collagen fibres. B. Odor~tohlasts. There were no apparent differences in the fine structure of control, fluorotic and recovery odontoblasts. At the level of resolution of the electron microscope. the cells seemed to be morphologically unaffected by fluoride. Predentine also appeared similar in the three groups.
Examination of dentine formation in rat DISCUSSION Method of jluoriae
administration
As discovered in our laboratory, a single subcutaneous injection of a high dose of sodium fluoride caused a local !severe ulceration. Several of these lesions would seriously impair the health of the animal; therefore, the interperitoneal route was selected for multiple injections. The ion was administered twice daily at a 6 hr interval to prolong its effects. Microradiography Earlier investigations (Yaeger, 1963; Yaeger and Eisenmann. 1963, Osmanski and Yaeger. 1964; Eisenmann and Yaeger, 1972) demonstrated that a single subcutaneous injection of sodium fluoride consistently induces a double responsean external band of hypermineralization and an adjacent. internal band of hypomineralization. On microradiographs, we found the respon:se in mineralization patterns following multiple injections to be similar to the single injection in respect of the formation of a band of hypermineralization corresponding to 1 day’s injection (Figs. 1 and 2). The adjacent band of hypomineralization was seldom visible. These bands of hypomineralization were known to be present, as evidenced by examining the ul trastructure (Figs. 3 and 4). Perhaps the discrepancy !n the previous investigations using a single dose and the present investigation using multiple doses relates to the different modes of administration, i.e. fluoride absorption, activity and clearance may be more rapid following intraperitoneal than following subcutaneous injection. The normal density of the “recovery” dentine indicates that the efI?cts of fluoride were temporary and the cells were callable of resumption of normal dentine formation (Fig. 5). Electron microscopy Interestingly. in developing fluorotic dentine, electron micrographs (Figs. 3 and 4) presented a contradictory appearance to that of microradiographs. Hypermineralized (relative to normal dentine) areas were indiscernable, whereas hypomineralization was striking. In fact. the patches devoid of mineral occupied most of the area in the response zone and globules of crystals occupied proportionately less area in most sections. However, these globules must be relatively dense because, microradiographically. bands of hyperm.neralization occurred through these regions. The composite effect of a thick section (the equivalent of thousands of ultra-thin sections) used for microradiography is obviously a significant factor. Evidently enough dense dentine globules formed within the altered regions to produce layers of high relative density. Yaeger (1963) examined the fine structure of hypermineralized and hypomineralized zones and observed crystals in both components; the hypermineralized contained more ‘norganic crystals per unit than did normal or hypomineralized dentine. which had fewer, but larger crystals. These observations contrast with a later investigation (Eisenmann and Yaeger. 1972) and with the present study, in which the hypomineralized bands had Ioatches which contained few, if any, crystals. In addition, neither areas of hypermineraliza-
4x7
tion nor variations in crystal size were observed ultrastructurally. The ultrastructure of the “recovery” dentine (Fig. 5) confirmed the impression of microradiographs (Fig. 2) that normal-appearing dentine forms after fluoride injections are terminated. A similar increase in radiodensity was observed by other investigators (Osmanski and Yaeger, 1965; Yaeger, 1966). As observed on electron micrographs, globules of crystals increased in size with time. Comparing the relative mineral patterns in Figs. 3 and 4 with Figs. 5 and 6. it is obvious that the size of the unmineralized patches steadily decreased. even as they became buried. In confirmation, Eisenmann and Yaeger (1972) recently reported that soon after these hypomineralized regions form, they arc capable of substantial mineral uptake. Possible
mechanisms of fluoride
disturbance
A possibility is alteration of the organic matrix by the presence of fluoride in the hypomineralized areas. or the secretion of an altered organic matrix by affected odontoblasts. Matrix changes-following single injections of fluoride-were shown with autoradiography (Yaeger. 1963 Gady and Yaeger. 1965). However, a recent investigation (Eisenmann and Yaeger. 1972) demonstrated that the hypomineralized matrix contains abundant, apparently normal collagen fibres that are immediately capable of substantial mineral uptake in vitro. The present study also indicates that the matrix per se is not permanently altered by the fluoride ion because hypomineralized regions contained collagen fibrils which steadily accumulated mineral (Figs. 6 and 7). These observations do not, however, rule out the possibility of subtle chemical changes in the matrix, rendering it temporarily unmineralizable. These changes would be reversible, allowing gradual mineral uptake. A possible factor could be the disturbance of phosphoprotein synthesis by the odontoblasts and/ or transport of phosphoproteins to the mineralizing front. Phosphoprotein appears to be synthesized by the odontoblast and has been reported (Weinstock and Leblond, 1973; Butler. Finch and Desteno. 1973) to constitute approximately 10 per cent of rat incisor dentine. This substance may serve as nucleation centres for initial deposition of crystals (Veis and Perry. 1967; Weinstock and Leblond. 1973). Relative to matrix alterations, some interesting comparisons can be made of the effects of fluoride on amelogenesis and dentinogenesis. Enamel and associated cells were taken from the same animals, as reported in a previous study (Walton and Eisenmann. 1974). Although the formative ameloblast manifests some structural alterations while under the influence of fluoride, it continues to secrete enamel matrix. Portions of the matrix are altered and do not acquire the normal number or demonstrate the customary uniform orientation of crystals. An area of fluoride response in enamel is visible in Fig. 6. In contrast, the odontoblast demonstrates no structural alterations. nor does it secrete a matrix which fails to mineralize with time. Another interesting possibility is the inhibition by fluoride of the mechanism which removes inorganic pyrophosphate from the matrix. This substance is present in high concentrations in dentine matrix
Examination
of dentin- frJrmation in rat
A.O.B.
f.p. 488
R. E. Walton and D. R. Eisenmann
Plate 2. Fig. 5. Electron micrograph of recovery dentine. This region corresponds to response zone seen in Fig. 2. The lastformed fluoride-response band is evident. An increasing uptake of mineral has eliminated most of the hypomineralized zone (0); the diminished unmineralized patches have persisted adjacent to the odontoblast processes (OP). Hypermineralization is not evident. Normal-appearing dentine (RD) has formed in the 3 days after cessation of fluoride injections. The mineralizing front (MF) appears normal. Ptedentine (PR). x2600 Fig. 6. A specimen taken from a recovery animal. An early-formed fluoride response zone lies near the dentineenamel junction (DEJ). The hypomineralized component persists as scattered patches largely devoid of crystals. Zones of hypomineralization (0) are evident in the overlying enamel (E). The bands in the enamel formed in response to fluoride administration concurrent to the formation of bands of hypomineralization in dentine (D). x 4000 Fig. 7. Higher magnification of the large unminera!ized patch in the dentine in Fig. 6. It consists primarily of collagen fibres with a similar arrangement and density to predentine. At the periphery, crystals overlie collagen profile (arrows). This patch also abuts an odontoblast process (OP); a portion is cut in tangential section. x 24,600
488
R. E. Walton
and D. R. Eisenmann
(Bisaz. Russell and Fleisch. 1968). It has been speculated (Fleisch and Russell, 1970; Woltgens, Bonting. and Bijvoet, 1971) that inorganic pyrophosphate must be locally destroyed by enzymes, such as pyrophosphatases or alkaline phosphatases, before dental mineralization can occur. The presence of fluoride. a potent inhibitor of both enzyme systems (Graham. Russell and Fleisch, 1970). could temporarily prevent the enzymes from removing pyrophosphate and thereby inhibit calcification. As demonstrated in recovery specimens (Figs. 2 and 5). the levels of fluoride were not sufficiently toxic to damage or permanently alter odontoblast function. therefore altered patterns of the hypomineralization were temporary and resumed when fluoride was cleared. Ackrlo~~lrdgrmolrs-The contributions of time. patience and technical assistance by the staff of the Department of Histology. and in particular Mrs. Elena Baltrusaitis, is greatly appreciated. The research was supported by NIDR Grant DE 03312.
REFERENCES
Bisaz S., Russell R. G. G. and Fleisch H. 1968. Isolation of inorganic pyrophosphate from bovine and human teeth. Arch ord Biol. 13, 6X3-696. Butler W. T.. Finch J. E. and Desteno C. V. 1972. Chemical character of proteins in rat incisors. Biochrm. Biophys. Artn 251, 167-171. Eisenmann D. R. and Yaeger J. A. 1969. Alterations in the formation of rat dentine and enamel induced by various ions. Arc/is orul Bid. 14, 104551064. Eisenmann D. R. and Yaeger J. A. 1972. In vitro mineralization of hypomineralized dentine induced by strontium and fluoride in the rat. .4rchs orul Biol. 17, 9X7-999. Fleisch H. and Russell R. G. G. 1970. Pyrophosphate and polyphosphate. In: I~~tcrruztiorul Encycloprdiu of Pharmacoloyy ud Thcrqwutics pp. 6lIIOO. Pergamon Press. Oxford.
Grady J. E. and Yaeger J. A. 1965. Polarizing microscopy of abnormal dentine produced by injections of strontium or fluoride. Arch oral Biol. 10. 175- 178. Graham R.. Russell G. and Fleisch H. 1970. Inorganic pyrophosphate and pyrophosphatases in calcification. and calcium homeostasis. C/in. Orthop. 69, 101-l 17. Kruger B. J. 1967. Histologic effects’of fluoride and molybdenum on developing dental tissues. Amt. dent J. 12. 5460. Osmanski C. P. and Yaeger J. A. 1964. Microradiography of rat incisor dentine during the development of the responsc to injected fuoride. hat. Rrc. 148. 467-483. Osmanski C. P. and Yaegcr J. A. 1965. The effect of parenteral Ruoride on rat incisor dentine as studied with microradiography. J. Orul Tlier. 1, 650. Schour 1. and Smith M. C. 1934. The histologic changes in the enamel and dentine of the rat incisor in acute and chronic experimental fluorosis. Liaitl. of Ark. Coil. .4yric., Ayric. Ezp. Stu.. Tech Bull. 52, 67-9 1. Veis A. and Perry A. 1967. The phosphoprotein of the dentine matrix. Biochmistr?: 6, 7409-2415. Walton R. E. and Eisenmann D. R. 1974. Ultrastructural examination of various stages of amelogenesis following parenteral fuoride administration. Arch oral Biol. 19. 171&182. Weinstock M. and Leblond C. P. 1973. Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentine of the rat incisor. /. Cell Biol. 56, 838845. Woltgens J. H., Bonting S. L.. Bijvoet 0. L. 1971. Relationship between alkaline phosphatase. inorganic pyrophosphatdse and L-ascorbic acid in calcifying hamster molars. C/in. Orthop. 78, 296301. Yaeger J. A. 1963. Microscopy of the response of rodent dentin to injected Houride. Anut. Rec. 145, 139-147. Yaeger J. A. 1966. Recovery of rat incisor dentine from abnormaI mineralization produced by strontium and huoride. tlnut. Rec. 154, 661-674. Yaeger J. A. and Eisenmann D. R. 1963. Response in rat incisor dentine to injected strontium, fluoride and parathyroid extract. J. dmt. Rex 42, 120881216. Yaeger J. A., Hinrichsen C. F. L. and Cohen M. J. 1964. Development of the response in rat incisor dentine to injected strontium and fluoride. Am. J. Amt. 114. 255271.
Fig. I. Microradiograph of a cross-section of an incisor from a fluorotic rat which received daily injections of sodium fluoride for 5 days and was killed on the 5th day. Four fully developed concentric bands of hypercalcification encompass the pulp space (P). (Insert) A narrow inner band of hypercalcification (arrow) is developing at the mineralizing front. Zones of hypomineralization are not evident in the area between the bands of increased density. Labial cap of enamel (E). Dentine formed prior to fluoride injections (D). Differences in radiodensity between mesial (M) and distal (Ds) components relate to differences in the gross thicknesses of the ground section. x 80 Fig. 2. Microradiograph of an incisor from a recovery animal. The four concentric bands of hypermineralization persist but are buried under a layer of dentine of normal-appearing density (insert). This recovery dentine (RD) adjacent to the pulp space was formed during the 3 days following the last administration of fluoride. x 80 Fig. 3. Electron micrograph of the mineralizing front of fluorotic dentine. This region corresponds to the response zone of Fig. 1. Pre-injection dentine (D) exhibits normal density. The region which was mineralizing at the time of fluoride administration demonstrates an erratic pattern of mineral uptake. Globules of dentine form but fail to coalesce to fill in and obscure the underlying predentine. Compare to the normal mineralizing front as demonstrated in Fig. 5. The linear pattern of the unmineralized regions is noticeable but not prominent, neither is the hypermineralized component (obvious on microradiographs) visible. Predentine (PR). Odontoblast process (OP). x 2400 Fig. 4. This region also corresponds to the response zone of Fig. 1. Occasionally areas are encountered where the unmineralized patches are scattered at random, demonstrating no linearity. x 2400