Uwe Kierdorf

Disturbances of the secretory stage of amelogenesis in fluorosed deer teeth: a scanning electron-microscopic study

Abstract.Structural changes resulting from fluoride-induced disturbances of the secretory stage of amelogenesis were studied in fluorosed dental enamel of ten permanent premolars and molars from roe deer (Capreolus capreolus) and red deer (Cervus elaphus). The fluorosed enamel exhibited surface hypoplasias of different depths and extents and an associated loss of its normal prism/interprism structure. The occurrence of such aprismatic enamel either was restricted to grossly accentuated and hypomineralized incremental (calciotraumatic) bands or affected more extended areas to the bottom of the hypoplastic lesions. The fluoride-induced disturbance of the secretory functions of the cells had thus been either temporary or permanent. Layers of aprismatic enamel were regarded as denoting periods of reduced enamel matrix formation by secretory ameloblasts lacking the distal, i.e., the prism-forming, portions of their Tomes processes. Our observations also indicated that the transition from the presecretory to the secretory stage of amelogenesis could be affected by fluoride, thereby preventing the ameloblasts from achieving their normal secretory function and from establishing fully formed Tomes processes. Aprismatic enamel was formed throughout the secretory stage of amelogenesis at these locations. The most severe ameloblast reaction that could be deduced from our findings was an abrupt cessation of enamel matrix secretion. Some of the pathological changes observed in fluorosed deer enamel showed striking similarities to those reported in rodents after acute parenteral fluoride dosing. Thus, periods of especially elevated plasma-fluoride levels in chronically fluoride-stressed deer can cause a disruption in the function of secretory ameloblasts similar to that following acute fluoride dosing in rodents.

Disturbed Enamel Formation in Wild Boars (Sus scrofa L.) From Fluoride Polluted Areas in Central Europe

The pathological alterations of enamel structure in the teeth of wild boars from fluoride polluted areas in N‐Bohemia (Czech Republic) and S‐Saxony (Germany) were studied on a macroscopic and a microscopic level. Mandibular bone fluoride concentration (mg F−/kg, dry wt; mean ± SD, individuals <24 months of age) in the specimens from N‐Bohemia (754.3 ± 149.6) and S‐Saxony (490.8 ± 135.1) was significantly higher than that of controls (free of dental fluorosis), originating from the western part of Germany (304.7 ± 91.0). Fluoride content in bulk enamel (mg F−/kg, ash wt) of fluorotic permanent teeth from N‐Bohemia (382.1 ± 165.2) and S‐Saxony (125.0 ± 38.3) was likewise significantly increased over that of non‐fluorotic control teeth from W‐Germany (33.6 ± 26.7). Macroscopically, fluorosed wild boar enamel exhibited opacity and discoloration of varying extent, accentuated perikymata as well as hypoplastic and posteruptive surface defects. Microradiographic and scanning electron microscopic analyses revealed enamel subsurface hypomineralization, accentuated Retzius lines and occurrence of broad, hypomineralized incremental bands of abnormal structure underlying hypoplastic enamel surface defects. The presence of zones of aprismatic enamel was associated with these bands. Incremental bands with altered enamel structure and enamel surface hypoplasias, both denoting a severe disturbance during the secretory stage of amelogenesis, have previously been observed in rodents following acute parenteral fluoride dosing. It is concluded that in the chronically fluoride exposed wild boars periods of especially elevated plasma fluoride levels exerted an acute toxic effect on the secretory ameloblasts. A feature not previously reported from fluorosed enamel was the occurrence of canal‐like structures that originated at the broad incremental bands and extended into the external enamel. The presence of these canals presumably results from a delay in the resumption of secretory activity by groups of ameloblasts following a fluoride insult. Based on experimental evidence in domestic pigs and in sheep, the overall subsurface hypomineralization of fluorosed wild boar enamel is attributed to a disturbance of enamel maturation. The distribution of fluorotic enamel changes within the dentition of the wild boars could be related to the developmental sequence of tooth formation in the species. Teeth whose crown formation took place prenatally (deciduous teeth) or largely pre‐weaning (permanent first molars) exhibited no or only moderate fluorotic enamel alterations. Based on the extension of enamel surface hypoplasias along the coronoapical axes of the tooth crowns, the timing of excess fluoride exposure that caused a marked disruption of enamel matrix secretion was estimated in specimens with a known date of death. The results indicate that the wild boars had been exposed to a particularly severe fluoride impact during autumn and winter of their first year of life. Anat Rec 259:12–24, 2000.

Mandibular bone fluoride levels and occurrence of fluoride induced dental lesions in populations of wild red deer (Cervus elaphus) from Central Europe.

Mandibular bone fluoride concentrations as well as frequency and intensity of fluoride-induced dental lesions were studied in four red deer populations from Czechia and Germany exposed to different levels of environmental fluoride. The degree of fluorosis in the permanent cheek teeth was recorded by a scoring system and the individual mandibles assigned to one of three categories (unfluorosed, slightly to moderately fluorosed, and markedly to severely fluorosed) on the basis of the tooth scores. An increase in the frequency and severity of dental fluorosis was positively associated with an increase in bone fluoride level, which itself was significantly different between the four samples. In all samples, the rate of bone fluoride accumulation was higher in younger than in older deer. The results of this study show that frequency and intensity of dental fluorosis can be used as valid indicators of increased fluoride exposure of deer populations, and thus as a quick, easy, economic and reliable means of monitoring fluoride pollution in areas inhabited by wild deer.

PATHOLOGICAL BONE CHANGES IN THE MANDIBLES OF WILD RED DEER (CERVUS ELAPHUS L.) EXPOSED TO HIGH ENVIRONMENTAL LEVELS OF FLUORIDE

A macroscopic, microscopic and scanning electron microscope study was performed on the pathological bone changes of the mandibles of wild red deer (n=61) exhibiting severe dental fluorosis. The animals originated from a highly fluoride polluted area in Central Europe (Ore mountains and their southern foreland, Czech‐German border region) and constituted 11.2% of the studied red deer sample (n=545) from this area. Pathologically increased wear and fracture of fluorosed teeth caused a variety of mandibular bone alterations, including periodontal breakdown, periostitis, osteitis and chronic osteomyelitis. As a further consequence of severe dental attrition, opening of the pulp chamber and formation of periapical abscesses were occasionally observed. In case of severe periodontal breakdown, loss of teeth from the mandibles was found. In addition to the inflammatory bone changes, the occurrence of osteofluorotic alterations was also diagnosed in the specimens with the highest bone fluoride concentrations (>4000 mg F−/kg dry wt). These changes comprised extended apposition of periosteal bone onto the mandibular cortex as well as deformation of the mandibular body, which was attributed to a fluoride‐induced osteomalacia. The present study provided circumstantial evidence that, in addition to fluoride induced dental lesions, the occurrence of marked periodontal disease and tooth loss is an important factor responsible for a reduction of life expectancy in severely fluorotic wild red deer.

State of Determination of the Antlerogenic Tissues with Special Reference to Double-head Formation

The developmental conditions for the growth of primary cranial appendages and secondary or subsequent antlers are discussed with special regard to ortho- and heterotopic double-head formation. We hypothesize that the cells of the pedicle periosteum, being derivatives of the initial or antlerogenic periosteum which is determined for the formation of primary cranial appendages, are equally endowed with special antlerogenic tendencies. The hormonally controlled activation of these tendencies is possible even if previous casting of the old antlers has not taken place, and no casting wound arises at the top of the pedicle. The fact that antler regeneration occurs regardless of previous detachment of the old antlers indicates that the stimulus for regeneration is not antler casting but the death of the velvet antler. Antler regeneration, like sequestration of the old antler, is thus hormonally suppressed for some months after velvet shedding.

Antlers as biomonitors of environmental pollution by lead and fluoride: A review

Antlers are periodically cast and re-grown cranial appendages of deer. Both endochondral and intramembranous ossification are involved in the formation of antler bone. After velvet shedding, antlers are completely bony structures which are referred to as hard antlers. Growing antlers accumulate substances with an affinity to mineralized tissues. Among these substances are lead and fluoride. Due to the seasonally limited life span of antlers, the concentrations of lead and fluoride in hard antlers reflect uptake by the forming bone during a defined, species-specific period of some months. Antlers can thus be viewed as “naturally standardized” environmental samples that are well suited as biomonitors of environmental pollution by bone-seeking contaminants. Because hard antlers are collected by hunters as trophies and kept in private or public collections, material for study can be obtained rather easily. So far, lead concentrations in hard antlers have been reported only from Europe, whereas data for fluoride are available from both Europe and North America. Some studies compared contaminant concentrations in antlers from different regions, while others analyzed time-trends in contaminant levels in antlers from a single region. Using the latter approach, a pronounced drop of lead concentrations during recent decades has been reported for antlers from various European countries. This indicates a marked decline of environmental lead levels that can be attributed mainly to the phase-out of leaded gasoline and, in addition, to a reduction of lead emissions also from stationary sources. In Germany, a corresponding drop has also been recorded for fluoride concentrations in antlers, which is attributed to a decline of fluoride emissions from stationary sources due to improved emission-control measures. There is some evidence that exposure to higher levels of fluoride may lead to an impaired antler mineralization. Using antlers as biomonitors has been shown to be an efficient method for assessing environmental pollution by lead and fluoride at low cost. Further studies using this now well established approach are therefore encouraged.

A quantitative backscattered electron imaging study of hypomineralization and hypoplasia in fluorosed dental enamel of deer.

Mineral content and distribution of fluorosed and unfluorosed (control) dental enamel of roe deer and red deer cheek teeth were analyzed using digital backscattered electron (BSE) imaging of PMMA-embedded specimens. Compared to the controls, the fluorosed enamel exhibited various aberrations resulting from a fluoride-induced disturbance of the processes involved in enamel formation. Thus, the presence of surface hypoplasias and an enhancement of the incremental pattern in the fluorosed enamel are evidence of a fluoride impact on the secretory ameloblasts, whereas a (subsurface) hypomineralization of different depth and extent is indicative of a fluoride effect on the maturation stage of amelogenesis. The marked variation in the severity of enamel hypomineralization seen along the coronocervical axis of a specimen pointed to a fluoride impact of varying intensity during this period of tooth development. Our observations further indicated that, in some locations, ameloblasts severely affected by fluoride during enamel matrix formation were able to recover from this insult and to function quite normally during the maturation stage of amelogenesis. A major advantage of the BSE imaging technique used in the present study over other methods is that it allows for a combination of micromorphological information with quantitative data on the mineralization of the analyzed tissue, which proved to be very useful for the characterization of fluoride-induced changes in dental enamel.

Monitoring regional fluoride pollution in the Saxonian Ore mountains (Germany) using the biomarker dental fluorosis in roe deer (Capreolus capreolus L.).

The spatial variation of fluoride contamination in the Saxonion part of the Ore mountains (Federal state of Saxony, Germany) was assessed by analysing the prevalence and severity of dental fluorosis in wild male roe deer aged 2 years or more (n = 794) from this region. The study area is exposed to increased atmospheric deposition of fluorides, originating from emission sources in the North-Bohemian brown coal belt. The severity of dental fluorosis in the permanent premolars and molars of one hemimandible per animal was assessed based on a scoring system and a dental lesion index of fluorosis (DLI) assigned to each specimen. On the basis of the mean DLI calculated for the roe deer originating from a certain municipal district, the respective deer sample was assigned to one of seven fluorosis categories, and a map showing the distribution of these categories was produced. Highest fluorosis categories were recorded for roe deer samples from the central and eastern parts of the study area that directly bordered on, or were located near to, the Czech-German border. A close spatial relationship between the main fluoride emission sources in North-Bohemia and the regions with the highest prevalence and severity of dental fluorosis in roe deer was discernible. The observed regional variation of fluoride pollution is in accordance with the results of other studies directly measuring fluoride deposition into the study area or fluoride levels in vegetation. A main advantage of the method used in this study over other ways of recording fluoride contamination of wildlife habitats is that it uses an established and continuously operated system of specimen collection, thereby enabling efficient monitoring with high spatial and temporal resolution at very low cost. Moreover, a clearly recognisable and relevant biological effect is measured which occurs in a dose-dependent manner over a range of environmentally relevant concentrations.

Treatment of the growing pedicle with retinoic acid increased the size of first antlers in fallow deer (Dama dama L.)

Unilateral injection of 10 mg of all-trans retinoic acid (RA) into the lateral portion of the growing pedicle of fallow bucks (n = 20) led to a significant (P = 0.033, Wilcoxon matched-pairs test) increase in first antler volume (median, 25.5 ml) as compared to the contralateral (control) side, injected with vehicle only (median, 21.5 ml). It is hypothesized that the RA treatment of the developing pedicle exercised a direct or indirect effect on the periosteal/perichondrial cells covering the growing cranial appendage, resulting in an increased proliferation rate of the cells of the antler perichondrium.

Histochemical and ultrastructural studies of cartilage resorption and acid phosphatase activity during antler growth in fallow deer (Dama dama)

Cartilage resorption in forming primary fallow deer antlers was studied by histochemistry and electron microscopy. A high activity of tartrate‐resistant acid phosphatase (TRAP), a histochemical marker of skeletal resorbing cells, was first detected in cells located in the mesenchymal tissue separating the columns of hypertrophic cartilage. No cartilage resorption was observed in this region. Intense TRAP staining occurred in large multinucleated cells (identified as inactive osteoclasts) as well as in smaller cells (regarded as mononuclear osteoclast progenitors). On the basis of these findings it was concluded that this was the region where osteoclasts differentiated from progenitor cells. Further proximally, the mineralized cartilage was eroded by active osteoclasts that were located in Howships lacunae and exhibited an intense TRAP staining. Electron microscopy showed that the cells identified as inactive osteoclasts lacked a polarized organization. In contrast, the active osteoclasts in the zone of cartilage resorption exhibited a typical polarized organization: the nuclei congregated near the basolateral cell surface, and there was a zone of deep membrane infoldings (ruffled border) surrounded by a clear zone at the apical cell pole adjacent to the resorption surface of the mineralized cartilage. The multinucleated cartilage‐resorbing cells of the forming antler thus exhibited the typical histochemical and morphological features of active mammalian osteoclasts. Low levels of TRAP activity were also observed in hypertrophic chondrocytes; however, the specificity and potential significance of this staining remain to be elucidated. Anat Rec 268:66–72, 2002.