Fluoride-induced alterations of enamel structure: an experimental study in the miniature pig.

We studied the structural changes in the enamel of mandibular third molars of miniature pigs administered a daily oral dose of 2 mg NaF (approximately 0.9 mg of fluoride) per kg body weight (added to the feed) for 1 year. The treatment period covered most of the secretory stage and the entire post-secretory stage of amelogenesis of the M(3). The enamel of the molars from the fluoride-fed pigs appeared opaque and chalky, and the erupted portions were stained brown. The underlying histopathological change was a pronounced subsurface hypomineralization of the enamel beneath a thin surface rim of higher mineral content. This enamel hypomineralization was attributed to a fluoride-induced impairment of the process of enamel maturation. The most conspicuous finding in the fluorotic enamel was the presence of numerous pit-type hypoplastic defects, denoting a marked fluoride-induced disturbance also of the secretory stage of amelogenesis. Microradiography and scanning electron microscopy revealed an enhanced incremental pattern in the outer enamel of the fluorotic molars. Typically, the bottom of larger hypoplastic defects was underlain by a broad, grossly accentuated incremental line. Occurrence of larger hypoplasias was further associated with the presence of aprismatic enamel, the formation of which was attributed to a loss of the prism-forming (distal) portion of the Tomes' processes of secretory ameloblasts. The findings in the miniature pigs closely parallel earlier observations on fluorotic enamel of free-ranging deer and wild boar from fluoride-polluted areas.

Age-related and site-specific changes in the pulpodentinal morphology of rat molars.

The rat molars are frequently used as experimental models in endodontic research, but there is little systematic information available on the influence of age on the pulpodentinal organ in Wistar rat molars and it is often difficult to evaluate more subtle changes following experimental interventions. The aim here was to describe changes with age in first upper Wistar rat molars with specific reference to the pulpodentinal organ. Animals were perfused with glutaraldehyde at 19 days, 1, 3, 6, 8, 12, 16, or 24 months of age. First upper molars from 56 animals were demineralized in EDTA, embedded in Epon, and processed for light and transmission electron microscopy. Substantial variation in the structure of the dentine and odontoblasts was observed within the root canals and the coronal pulp chamber. In general, odontoblasts changed from a tall, columnar morphology in the coronal pulp chamber to a more cuboidal or flattened shape near the apex, particularly towards the interradicular space. Secondary dentine formation was more pronounced along the mesial aspect of the root chamber and corresponding to the bottom of fissures. Local tertiary dentine formation was layered in the upper pulp chamber, corresponding to occlusal attrition of the cusp. In several molars a local formation of irregular tertiary dentine was observed cervically in the mesial pulp chamber. After 1 year, a distinct protrusion of irregular dentine extended into the mesiocervical pulp, apparently corresponding to a denudation of cervical root dentine. Experimental pulp-capping studies frequently use first upper rat molars with perforations made through the mesial aspect of the crown; such perforations might be close to the irregular dentine in the mesiocervical region. In conclusion, this study identifies age-associated and regional changes of pulpodentinal morphology in first upper rat molars. Therefore, evaluation of morphological alterations following vital-pulp experiments should be done in specific age groups and at specific sites in the pulp.

The influence of corneal stromal matrix proteins on the migration of human corneal fibroblasts.

Motivated by the alterations seen in the corneal matrix composition after photorefractive keratectomy and the migration of corneal keratocytes seen following this procedure, the locomotor response of corneal stromal fibroblasts to various extracellular matrix proteins was determined. In addition, the involvement of integrin mediated attachment to the matrix proteins was investigated. Quantitative invasion assays were performed using collagen gels, supplemented with either fibronectin, tenascin, collagen type V, collagen type VI, chondroitin sulfate or keratan sulfate. The ultrastructure of the gels was visualized by scanning electron microscopy and related to the migration results. The extent of alpha(1)beta(1), alpha(2)beta(1), alpha(3)beta(1)and alpha(5)beta(1)integrin mediated attachment to the matrix proteins was evaluated using blocking antibodies. Fibronectin increased corneal fibroblast migration significantly, and served as an excellent substrate for cellular attachment, mediated by the alpha(5)beta(1)integrin. Addition of tenascin to the fibronectin-containing gels disrupted these effects, while attachment to this matrix also involved the integrins alpha(2)beta(1)and alpha(3)beta(1). Chondroitin sulfate and collagen types V and VI primarily altered the structure of the collagen matrix, resulting in an inhibition of migration by the collagens and an increase by chondroitin sulfate. They all served as poor substrates for attachment. Thus, the migratory activity of corneal fibroblasts in vitro is influenced by the composition of the surrounding extracellular matrix, either by integrin mediated cell-matrix interactions or through matrix-matrix interactions. This study provides evidence that the provisional matrix deposited in a corneal stromal wound may facilitate the entry of migrating corneal fibroblasts.

Selective but nonspecific immunolabeling of enamel protein-associated compartments by a monoclonal antibody against vimentin.

Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues. (J Histochem Cytochem 47:1237-1245, 1999)

Resorption of hydroxyapatite and fluorapatite ceramic coatings on weight-bearing implants: a quantitative and morphological study in dogs.

Resorption (defined as loss of ceramic coating because of cellular activity or dissolution) of ceramic coatings is a matter of concern for the long-term performance of ceramic-coated implants. A new fluorine-containing coating, fluorapatite (FA), has been shown to be more stable than hydroxyapatite (HA) in unloaded models. In a weight-bearing model in trabecular bone, we evaluated loss (defined as reduction of coating irrespective of type of mechanism) of HA and FA coatings during 25 weeks of implantation. Eight mature dogs had HA- or FA-coated implants inserted bilaterally into the weight-bearing region of the medial femoral condyle. Quantified loss of ceramic coating was estimated at the light microscopic level using stereological methods. The experiment showed significant loss of both types of coatings. However, no statistical difference in loss of ceramic coating was found regarding surface area implant coverage, volume, and thickness (p = 0.77, p = 0.13, p = 0.56, p = 0.23, respectively). Completely resorbed HA coating was replaced by 36 +/- 6.0% (range: 26-42) bone in direct contact with the implant surface compared with 29 +/- 16.0% (range: 12-59) for FA (p = 0.40), suggesting that the implant was firmly fixed despite loss of the ceramic coating. Transmission electron microscopy in combination with electron energy spectroscopy and electron spectroscopic imaging showed that osteclast-like cells, osteocytes, macrophage-like cells, and fibroblasts had phagocytosed calcium-containing fragments, indicating cell-mediated resorption of the ceramic coating.

Role of different loading conditions on resorption of hydroxyapatite coating evaluated by histomorphometric and stereological methods.

The role of different loading conditions on resorption of plasma-sprayed hydroxyapatite coating was investigated in an experimental study. Resorption of hydroxyapatite was quantified by histomorphometric and stereological methods on backscattered scanning electron images. Hydroxyapatite-coated titanium implants were inserted unilaterally into the medial femoral condyle of the knee in 14 mature dogs. Initially, all implants were subjected to controlled micromotion of 150 microns. After 4 weeks, the dogs were randomly assigned either to have the implant surgically immobilized to prevent further micromovement or to have a sham operation. Sixteen weeks after the first operation, the implants were analyzed. Six noninserted implants served as controls. The surface area and volume of the hydroxyapatite coating were reduced on the immobilized implants by 53 and 67% (p < 0.05), respectively, and were further significantly reduced on the continuously loaded implants by 83 and 87%, respectively, compared with the control implants. The hydroxyapatite coating was significantly thinner on immobilized (15 microns) and continuously loaded (15 microns) implants as compared with control implants (23 microns), but no difference between the inserted implants was found. Areas not covered with hydroxyapatite had 29 and 24% bone coverage on the immobilized and continuously loaded implants (not significant). Resorption of hydroxyapatite coating did occur in vivo. Continuous loading of the implants accelerated resorption significantly compared with immobilization of the implants. It is suggested that completely resorbed hydroxyapatite was partly replaced by bone in direct contact with the metal implant surface.

Degradation of the dental basement membrane during mouse tooth development in vitro.

During tooth development, the basement membrane is degraded at the late bell stage, but the developmental significance of this event is not known. Organ culture offers a method where developmental processes can be manipulated in controlled conditions. We cultured bell-stage tooth germs either in a chemically defined or a serum-containing medium and analyzed the degradation of the basement membrane by different methods. Type IV collagen was present throughout the dental basement membranes at the epithelial-mesenchymal interface at the onset of culture. After 10 days of culture, irrespective of the medium used, type IV collagen and laminin had disappeared from the cuspal areas but were present at the cervical loop. As was the case in vivo, the expression of 72 kDa type IV collagenase gene was intense in the differentiating preodontoblasts and in the odontoblasts during secretion of the first predentin matrix near the cuspal tips. Ultrastructural observations showed that the basal lamina had been removed in all cultured tooth organs. Also, the breakdown of the basement membrane occurred irrespective of the presence of mineral in the dentin matrix. Our observations suggest in contrast to earlier observations, that there are no major differences in basic events leading to dentino- and amelogenesis, when tooth organs are cultured in the presence or absence of serum.

Scanning electron microscopy of final enamel formation in rat mandibular incisors following single injections of 1-hydroxyethylidene-1,1-bisphosphonate.

A single, high dose of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) results in three different types of lesions along the enamel surface of the rat incisor, one of which is seen as a "bright band" crossing the final enamel surface in the scanning electron microscope (SEM). The present study presents the structural surface features of final enamel formation and its subsequent maturation in normal and HEBP-exposed rats. The position of the bright band is examined in relation to where the Tomes processes pits disappear (DTPP), where the boundary between "light" and "dark" enamel (LDB) as seen by SEM is located, and in particular, where the so-called opaque boundary (OB) is positioned. Groups of rats were given a subcutaneous dose of 0, 5, or 10 mg P/kg body wt of HEBP and killed at intervals of either 12 hours or 2 or 9 days. The mandibular incisors were processed for SEM after enzymatic digestion of enamel organ remains. Enamel surface nodules, 100-300 nm in diameter and composed of smaller units, were evident at the start of final enamel formation which was defined as the area from DTPP to LDB. With increasing maturation, the nodules merged to form a smooth surface. In HEBP-treated animals, growth and merging of these surface nodules became arrested at the time of injection resulting in an irreversible "porous" stage corresponding to that part of the surface enamel. This area--the bright band--developed corresponding to the start of the area of final enamel formation and was subsequently carried incisally during the eruption of the incisor.(ABSTRACT TRUNCATED AT 250 WORDS)

Tensile bond strength of resin-bonded non-precious alloys with chemically and mechanically roughened surfaces.

The present study was carried out for investigation of the tensile bond strength of resin-bonded non-precious alloys after their surfaces were roughened by sand-blasting, chemical etching, or sugar crystal impressions. Fifty test specimens were cast in a Ni-Cr (Wiron 88) alloy and 50 in a Co-Cr (Wirobond) alloy. Twenty specimens of each alloy were surface-treated according to the sugar crystal impression method. The remaining specimens were first sand-blasted, and 20 specimens of each alloy were thereafter allocated for chemical etching and divided into subgroups with different etching conditions. The samples were chemically etched in strong inorganic acid solutions. After being etched, the specimens were bonded together in pairs by a chemically-curing resin cement (Panavia EX) with a force of 2 kg/cm2. After cementation, the specimens were stored under humid conditions at 37 degrees C for three wk. Prior to being tested, the specimens were subjected to 1000 thermal cyclings at temperatures between 10 degrees C and 55 degrees C. The tensile bond strength tests showed that Ni-Cr specimens sand-blasted and thereafter etched with a 50% conc. of HNO3 and a 50% conc. of HCl for two min and Co-Cr specimens sand-blasted and etched (conc. HCl for 15 min or three h) or sand-blasted alone resulted in similar high bonding values ranging between 33.3 and 37.2 MPa. Surface roughening with use of the sugar crystal impression method resulted in statistically significant lower bond strength values for both alloys (Ni-Cr, 17.9 MPa; Co-Cr, 10.2 MPa).

Posteruptive changes in human dental fluorosis--a histological and ultrastructural study.

The aim of the present study was to describe the structural features characterizing the severe grades of human fluorotic enamel (TF scores 5-9) with particular emphasis on the posteruptive changes in severely fluorosed teeth. Dental fluorosis is a subsurface hypomineralized lesion deep to a well-mineralized outer enamel surface, which in severe cases breaks apart shortly after eruption. Early signs of posteruptive changes comprise small defects corresponding to the opening of striae of Retzius. The enamel pits which develop after eruption in more severe cases exhibit an increase in mineral content at their base which correspond to the exposed subsurface hypomineralized lesions. Likewise, the extensive removal of surface enamel in the most severe cases of human fluorosis results in a highly varying uptake of mineral into the exposed subsurface hypomineralized lesions. The uptake varies greatly within apparently similar degrees of hypomineralized lesions. In approximal abrasion facets, however, where the subsurface lesions are also exposed, no evidence of mineral uptake was found. At the ultrastructural level, the well-mineralized surface zone consists of large hexagonal enamel crystals separated by rather large intercrystalline spaces in which numerous irregular small crystals are observed. Moreover, the large crystals may exhibit central and peripheral dissolution. In addition, mineral appeared to be deposited into such defects as well as along the side of the crystals, often with the lattices being continuous from the original crystal into the apparently posteruptive formed crystal material. It is concluded that a substantial mineral uptake can take place in exposed porous hypomineralized fluorotic enamel after eruption, but is most likely to be associated with the presence of microbial deposits, the metabolic activity of which may play a keyrole in mineral exchange.

The effect of a single dose of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) on presecretory ameloblast differentiation in rat incisors.

A single, high dose of HEBP to rats results in a triad of lesions along the mineralizing front of the incisor enamel. One type of lesion is a shallow groove crossing the apical enamel surface. The purpose of this study was to explore the pathogenesis of this "demarcation groove", and to characterize changes in the involved regions of amelogenesis. Rats were given a subcutaneous dose of 10 mg/kg body weight of HEBP and sacrificed by vascular perfusion at intervals ranging from 1 to 36 hours. Mandibular incisors were processed for light and electron microscopy. The region of ameloblasts facing dentin was divided into two subregions: A region of ameloblasts facing unmineralized dentin, comprising a posterior (Aud/p) and an anterior portion (Aud/a), and a region of ameloblasts facing mineralized dentin (Amd). The progressive apical mineralization of the predentin was arrested up to 12 hours after injection of HEBP, while ameloblasts related to already mineralizing dentin continued to differentiate and secrete enamel matrix. At 8 hours the dentin and enamel layers had assumed a common apical border at the start of Amd, marking the position of the future demarcation groove. The length of Aud/p remained constant, Aud/a doubled in length, and Amd was drastically reduced up to 24 hours after injection of HEBP. The normal migration rate of the ameloblasts was unaffected by HEBP. Accumulations of ameloblast secretory products occurred at certain time intervals between the cell apices, but no morphological changes were recorded in the organelles. Most of the changes observed may be indirect in nature resulting from the physico-chemical effect of HEBP on normal mineralization of dentin and enamel. However, further studies are needed to elucidate possible direct cellular effects on ameloblasts.

The effect of a single dose of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) on secretory ameloblasts and enamel formation in rat incisors.

The present experiment was undertaken in order to study how HEBP affects secretory ameloblasts and the mineralizing front of rat incisor enamel resulting in the formation of a hypomineralized incremental band. Rats were given a single subcutaneous injection of 10 mg/kg body weight of HEBP and sacrificed at intervals ranging from 1 hour to 9 days. The incisors were specially prepared for microradiography, scanning electron microscopy, and light and transmission electron microscopy. The microradiographical examination 9 days after injection revealed a distinct incremental band of hypomineralization from the amelo-dentinal junction to the outer enamel surface. The overall rod pattern and mineral distribution within the enamel was otherwise normal. Under light microscopy the ameloblasts exhibited an increase in vacuoles and dark granules in the supranuclear cytoplasm 2-12 hours after injection. At 12 hours a total disarray of the mineralizing front was evident and confirmed by SEM. However, 24 hours after injection a normal structure of the mineralizing front was regained. Ultrastructurally the organelles of the ameloblasts showed no changes from normal at any time interval. However, 2 hours after injection multivesicular bodies appeared frequently in the Tomes' processes and crystal density had diminished in the interrod enamel. At 8 hours a proteinaceous matrixdevoid of crystals was accumulating corresponding to interrod growth regions, whereas no obvious changes were recorded at the rod growth regions. At 12 hours the newly formed interrod enamel was thin and without crystals in some places. In such areas a granular, less-dense matrix was found and a band of small swellings of the interrod enamel was evident most likely corresponding to the time of injection. After 24 hours the cells and the mineralizing front appeared normal except in the interrod growth regions where the cell processes were still separated by wide intercellular spaces in which crystals were occasionally found in a fine granular matrix. These observations are discussed with particular reference on the known physico-chemical effects of HEBP on mineral formation. A possible direct cellular effect on the ameloblasts should be studied using radioautography and immunocytological techniques.

Effects of single doses of 1-hydroxyethylidene-1,1-bisphosphonate on the mineralizing front of rat incisor enamel: a microradiographic and scanning electron microscopic study.

The effects of a single dose of HEBP were examined by injecting rats subcutaneously with various doses (0.5, 1, 2.5, 5 and 10 mg P/kg body wt) and killing them either 2 or 9 days after injection. The maxillary incisors were processed for microradiography and the mandibular incisors for scanning electron microscopy after enzymatic digestion of the enamel organ remains. All doses resulted in a hypomineralized incremental band corresponding to the position of the mineralizing front at the time of injection. At 5 and 10 mg P/kg body wt, a hypomineralized lesion was found throughout the whole thickness of the enamel in an area which corresponded to the stage of late enamel secretion at the time of injection. The surface layer covering this lesion was elevated or disrupted. By scanning electron microscopy, there were three different types of lesions along the enamel surface: a "demarcation groove" corresponding to the initial enamel formation at the time of injection; an "area containing mineral globules" and a "bright band" corresponding to the stages of late secretory and final enamel formation, respectively, at the time of injection. A single injection of HEBP thus interferes with different stages of enamel formation. The findings may be explained as of the physico-chemical effects of HEBP on the mineral phase alone, but a direct effect of the drug on ameloblast function cannot be excluded.

Maturation in developing permanent porcine enamel.

Mineral content per tissue volume was investigated in developing permanent porcine enamel and contrasted with weight-related data. Levels of mineralization were correlated directly with the histological appearance of the overlying enamel organ. Magnesium concentrations were measured at different stages of enamel development. Mineral levels rose from approximately 30% per volume of tissue during the secretory stage to approximately 60% in mature tissue. This is much lower than final mineral levels in enamel of other species. Enamel containing low mineral levels was adjacent to tall secretory ameloblasts which had reduced in height by approximately 50% at a point corresponding to the beginning of the maturation stage. Magnesium concentrations remained relatively constant throughout the secretory stage, at 0.2% Mg by weight. These rose by 3-4 times in the enamel of the maturation stage. The low levels of mineralization in the mature porcine enamel did not appear to be due to enamel pathology, and the possibility of porcine teeth erupting in an immature, partially porous condition is discussed.

Organic structures of developmental origin in human surface enamel.

The aim of this study was to examine the distribution of organic material in mature enamel surfaces immediately prior to eruption. Thirty-six samples of buccal or lingual enamel from unerupted third molars were prepared for transmission electron microscopy by a method involving demineralization of the enamel after embedding in Epon. The results showed that at time of eruption human surface enamel is a highly porous structure containing large amounts of developmental protein which appear as a variety of triangular, funnel-shaped, or invaginated configurations extending into the enamel. The implications of this finding may be of importance to the understanding of early caries lesion formation in the enamel since organic structures may modify the diffusion of ions in and out of the tissue.

Mineral and protein concentrations in enamel of the developing permanent porcine dentition.

Calcium, phosphorus and protein analyses have been performed on developing permanent enamel from the mandibular dentition of the domestic pig. The pattern of mineralization and protein loss was similar from tooth to tooth and similar to teeth from other species. Comparison of different teeth at the same developmental stages (secretion--stage 1, transition--stage 2 and maturation - stage 3) revealed remarkably similar concentrations of calcium, phosphorus, and protein regardless of tooth type. These data were similar to those from other deciduous dentitions, except that maturing/mature tissue in the pig seemed less well mineralized.

Cyclic localization of actin and its relationship to junctional complexes in maturation ameloblasts of the rat incisor.

The patterns of fluorescence associated with maturation ameloblasts of mandibular incisors labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) for the detection of F-actin were investigated in normal and fluoride-treated rats. In normal rats, bands of smooth-ended ameloblasts (SA) exhibited intense fluorescence at their proximal ends only. Bands of ruffle-ended ameloblasts (RA) exhibited strong fluorescence at their distal ends as well as at their proximal ends. Regional differences in degree of intensity within the bands and between bands were displayed. In the apical part of the RA bands the proximal fluorescence was intense; it then decreased in an incisal direction; and it finally was absent close to the adjacent SA band. The incisal extension of strong proximal fluorescence in RA bands was short in early maturation and long in late maturation. The fluorescence pattern at both ends of the ameloblasts was cyclically repeated throughout the region of ameloblast modulation corresponding to the numbers of SA bands. In rats receiving 113 ppm fluoride in their drinking water for 2 months the number of fluorescence and ameloblast modulation cycles was reduced equally indicating that the cyclic F-actin localization is a phenomenon related to ameloblast modulation. Electron microscopy revealed that areas of strong fluorescence contained filament bundles, presumably actin filaments, in relation to continuous junctions occluding the interameloblast spaces. Areas of weak or no fluorescence were related to discontinuous macular junctions. The results suggest that the changes in F-actin distribution correlate well with junctional complex development, and therefore, possible functions related to the intermeloblast spaces within the RA bands may be redistributed as the ameloblasts are carried incisally by the erupting incisor.

Developmental stages in permanent porcine enamel.

Developing permanent teeth of different ages were obtained from Danish Landrace pigs. Visibly distinct zones on their enamel surfaces were shown to correspond to changes in chemical composition previously reported for other species. The time of appearance, rate of progress and duration of each stage was determined for each tooth type.

Dental fluorosis developed in post-secretory enamel.

The aim of this study was to test whether dental fluorosis can be produced by administration of chronic doses of fluoride during only the post-secretory stage of enamel mineralization. Eight control and eight experimental pigs matched by weight and litter were fed a low-fluoride diet (less than 0.05 mg F-/kg b.w. daily) from weaning to slaughter at 14 months. The test group received an oral dose of 2 mg F-/kg b.w. per day from 8 months of age. Lower fourth pre-molars were at the post-secretory stage at the start of fluoride administration (confirmed by tetracycline marker) and were just erupting at slaughter. All of the fourth pre-molar teeth from the test group developed diffuse enamel hypomineralization indistinguishable from human fluorosis. No such lesions were seen in any of the teeth from the control animals. It was concluded that enamel fluorosis may be caused by fluoride exposure in the maturation phase only. The pathogenic mechanism may be an effect either on the selective loss of protein or on the influx of mineral, both of which occur during the post-secretory or maturation stage of enamel formation.