The initial process of enamel prism arrangement and its relation to the Hunter-Schreger bands in dog teeth

The three-dimensional architecture of enamel prisms at early stages of enamel formation and its spatial relationship to the Hunter-Schreger bands were examined in canine tooth germs by light and electron microscopy. In serial semithin sections of demineralized tooth germs tangential to the enamel-dentin junction, a straight row of enamel prisms was depicted along the longitudinal tooth axis at the level of the enamel-dentin junction and then their three-dimensional arrangement was reconstructed using computer software. The spatial arrangement of the groups of enamel rods oriented in specific sideward directions was also reconstructed in deep layers of the enamel. Initially, all enamel prisms were parallel to perpendicular toward the enamel-dentin junction, but at 10μm from the enamel-dentin junction, some small specks, or groups of enamel prisms--tilting to the right or the left--emerged as small islands. In each speck of enamel prism, the inclined prisms were uniformly oriented in a sideward direction and gradually expanded their boundary until merging with the neighboring specks inclined in the same direction. Consequently, at 50μm from the enamel-dentin junction, the group of enamel prisms oriented either to the right or the left formed alternately arranged horizontal belt-like zones, corresponding to the parazone or the diazone of the Hunter-Schreger bands. Reversed images of scanning electron-micrographs of the exposed surfaces of the developing enamel revealed round and bulb-like profiles of Tomes' processes at early amelogenesis and its changes into a characteristic structure combined with flat secretory and enclosing nonsecretory faces that dictated the orientation of corresponding enamel prisms. The results suggest that the groups of enamel prisms oriented in sideward directions first appear as small island-like specks near the enamel-dentin junction, which later merge and form alternating horizontal belt-like zones as a consequence of morphological changes of the Tomes' processes. However, the mechanisms whereby the functional grouping of secretory ameloblasts with similarly oriented Tomes' processes is induced are yet to be determined.     

Microscopy of the neonatal line in developing human enamel

The neonatal line in developing human primary teeth was examined by means of phase contrast microscopy, microradiography and transmission electron microscopy. When thin sections were observed by light microscopy, the lateral dimensions of the line were not as extensive as had been previously reported. The line had a “staircase” configuration and appeared to be identical to published light micrographs of the stria of Retzius. On radiograms, the lateral extent of the hypomineralization was also decreased. The ultrastructural basis for the neonatal line appeared to be a localized change in configuration of enamel prisms along with a possible reduction in crystal concentration. The possibility that some rods actually end at the line could not be excluded, however.     

Neurons containing retrogradely transported Fluoro-Gold exhibit a variety of lysosomal profiles: A combined brightfield, fluorescence, and electron microscopic study

Summary The advantages of axonally transported Fluoro-Gold as a retrograde fluorescent marker are numerous. The objective of the present study was to determine whether transported Fluoro-Gold is visible in either semi-thin sections for light microscopy or thin sections for electron microscopy. Rats received injections of Fluoro-Gold into either the striatum or thoracic spinal cord. After appropriate survival times, labelled neurons were observed with the fluorescence microscope in brain regions that are known to project to the injected areas.Sections that contained labelled cells were embedded in plastic and examined with a fluorescence microscope. Semi-thin sections of unosmicated tissue displayed high-resolution fluorescent labelling of somata and dendrites. In contrast, osmicated tissue did not fluoresce, but numerous dark granules were observed in the dendritic and perikaryal cytoplasm of labelled neurons in toluidine blue stained sections that were examined with brightfield optics. The unosmicated tissue did not display these granules, and this finding suggested that the granules are composed of membranes. Neurons in other brain regions that are known not to project to the injection sites did not contain these dark granules.Adjacent thin sections examined with the electron microscope displayed numerous electron-dense, lysosome-like organelles in the cytoplasm of labelled neurons. The electron density of these organelles was greater than that of lysosomes in unlabelled neurons. Three types of distinctive organelles were observed in these preparations: (1) relatively dense concentric lamellar bodies of various sizes; (2) heterogeneous or lipofuscin-like lysosomes; and (3) coarse grained lysosomes. Control sections and unlabelled neurons did not display these organelles. Therefore, these organelles appear to correlate with Fluoro-Gold localized within the somata and dendrites of retrogradely labelled neurons. It is not known if they are the Fluoro-Gold itself, or represent a physiological effect on membranes. The results of this study indicate that Fluoro-Gold may be useful for tract tracing at the electron microscopic level.     

Appositional enamel growth in molars of South African fossil hominids

Enamel is formed incrementally by the secretory activity of ameloblast cells. Variable stages of secretion result in the formation of structures known as cross striations along enamel prisms, for which experimental data demonstrate a correspondence with daily periods of secretion. Patterns of variation in this daily growth are important to understanding mechanisms of tooth formation and the development of enamel thickness. Transmitted light microscopy (TLM) of histological ground sections and scanning electron microscopy (SEM) of bulk specimens or their surface replicas are the usual methods for investigating cross striations. However, these methods pose some constraints on the study of these features in Plio-Pleistocene hominid enamel, the specimens of which may only rarely be sectioned for TLM or examined on only their most superficial surfaces for SEM. The recent development of portable confocal scanning optical microscopy (PCSOM) resolves some of the restrictions on fractured enamel surfaces, allowing the visualization of cross striations by direct examination. This technology has been applied here to the study of Australopithecus africanus and Paranthropus robustus hominid molars from the Plio-Pleistocene of South Africa. We hypothesize that these taxa have increased enamel appositional rates compared with modern humans, because despite having thicker enamelled molars (particularly P. robustus), the enamel crowns of these fossil taxa take an equivalent or reduced amount of time to form. Cross striations were measured in cuspal, lateral and cervical regions of the enamel crowns, and, within each region, the inner, middle and outer zones. Values obtained for A. africanus outer zones of the enamel crown are, in general, lower than those for P. robustus, indicating faster forming enamel in the latter, while both taxa show higher rates of enamel growth than modern humans and the African great apes. This demonstrates a relatively high degree of variability in the mechanisms underlying the development of enamel across taxa.     

Morphometry and Patterns of Lamellar Bone in Human Haversian Systems

The lamellar architecture of secondary osteons (Haversian systems) has been studied with scanning electron microscopy (SEM) in transverse sections of human cortical bone. Na₃PO₄ etching was used to improve the resolution of the interface between neighboring lamellae and the precision of measurements. These technical improvements permitted testing of earlier morphometry assumptions concerning lamellar thickness while revealing the existence of different lamellar patterns. The mean lamellar thickness was 9.0 ± 2.13 μm, thicker and with a wider range of variation with respect to earlier measurements. The number of lamellae showed a direct correlation with the lamellar bone area, and their thickness had a random distribution for osteonal size classes. The circular, concentrical pattern was the more frequently observed, but spiral and crescent‐moon‐shaped lamellae were also documented. Selected osteons were examined by either SEM or SEM combined with polarized light microscopy allowing comparisons of corresponding sectors of the osteon. The bright bands observed with polarized light corresponded to the grooves observed in etched sections by SEM. The dark bands corresponded to the lamellar surface with the cut fibrils oriented approximately longitudinally along the central canal axis. However, lamellae with large and blurred bright bands could be observed, which did not correspond to a groove observed by SEM. These findings are in contrast with the assumption that all the fibril layers within a lamella are oriented along a constant and unchangeable angle. The different lamellar patterns may be explained by the synchronous or staggered recruitment and activation of osteoblasts committed to the osteon's completion. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

The developmental clock of dental enamel: a test for the periodicity of prism cross-striations in modern humans and an evaluation of the most likely sources of error in histological studies of this kind

Dental tissues contain regular microscopic structures believed to result from periodic variations in the secretion of matrix by enamel- and dentine-forming cells. Counts of these structures are an important tool for reconstructing the chronology of dental development in both modern and fossil hominids. Most studies rely on the periodicity of the regular cross-banding that occurs along the long axis of enamel prisms. These prism cross-striations are widely thought to reflect a circadian rhythm of enamel matrix secretion and are generally regarded as representing daily increments of tissue. Previously, some researchers have argued against the circadian periodicity of these structures and questioned their use in reconstructing dental development. Here we tested the periodicity of enamel cross-striations – and the accuracy to which they can be used – in the developing permanent dentition of five children, excavated from a 19th century crypt in London, whose age-at-death was independently known. The interruption of crown formation by death was used to calibrate cross-striation counts. All five individuals produced counts that were strongly consistent with those expected from the independently known ages, taking into account the position of the neonatal line and factors of preservation. These results confirm that cross-striations do indeed reflect a circadian rhythm in enamel matrix secretion. They further validate their use in reconstructing dental development and in determining the age-at-death of the remains of children whose dentitions are still forming at the time of death. Significantly they identify the most likely source of error and the common difficulties encountered in histological studies of this kind.     

Organization of crystals in enamel

It has been variously suggested that the organic matrix associated with the mineral phase of enamel is present as either calcified fibrils, central dark lines, peripheral sheaths around hexagonal crystals, or organic ghosts apparently contained within crystal profiles. The most consistent findings confirm the crystal ghost conception. Grid decalcification of nearly mature sectioned enamel and staining revealed hollow, noncrystalline structures whose external measurements were statistically identical to those of the dissolved crystallites, but with internal measurements too small to accommodate the crystallites. To explain these apparent ghosts in view of the incompatibility of ghosts with crystal structure, it has been proposed that the crystallites are not hexagonal in cross-sections and the hexagonal appearance is due to projections of parallelepiped-shaped crystallite segments with cut surfaces that are rhomboidal in shape. Material on the surface of such profiles would project as if it were contained within the profile. Hexagonal forms could not be demonstrated in isolated crystallites examined by transmission electron microscopy, high-resolution scanning electron microscopy, and replicas made of the isolated crystallite preparations examined by transmission electron microscopy. Existing evidence does not rule out the possibility that the noncrystalline profiles represent stain drawn into the holes left by the dissolved crystallites as a result of high capillarity forces.     

Effects of KrF excimer laser irradiation on human dental enamel

Spectra of human dental enamel was recorded in the 200-400 nm UV region. It showed the weak band at 280 nm which were present in enamel protein. Excimer laser are gas lasers which emit light with photochemical decomposition. The wavelength depends on the 248 nm with krypton-fluoride. The enamel surfaces of extracted human teeth were exposed to KrF excimer laser by an energy density range from 29.6 to 3200 J/cm2. The purpose of this study was to investigate the changes with photo chemical reaction in enamel by light microscopy, SEM and X ray diffraction method. Results of analyses suggested that the observed changes of enamel exposed to this laser were the alpha and beta-tricalcium phosphate (TCP) phase in small amounts. No histological changes were observed in grain boundaries of cross sectioned lased enamel under light microscopy. The SEM examination revealed a roughened surface with bubble formation at 800-3200 J/cm2. SEM of etched enamel surface with 0.1 N HCl after laser irradiation at 400-800 J/cm2 showed the extension along the length of the rods. At 1600-3200 J/cm2, there appeared to be a melting of prism structures, because of conversion of photon energy into thermal energy. These results showed the KrF excimer laser irradiation to dental enamel might be a new type of treatment modality and diagnosis in preventive dentistry.     

Types of callosally projecting nonpyramidal neurons in rat visual cortex identified by lysosomal HRP retrograde labeling

Summary Callosally projecting neurons, labeled following injection of horseradish peroxidase (HRP) into the 17/18a border of the contralateral hemisphere, have been examined by light and electron microscopy. These neurons exhibit two types of horseradish peroxidase labeling: either a diffuse, Golgi-like labeling, or a granular, punctate labeling. The punctate type of HRP-labeling is the predominant form in nonpyramidal neurons, while pyramidal neurons frequently display either diffuse or punctate labeling. Only punctately labeled neurons have been examined in this study. Light microscopic analyses of 1-m sections show that in the heavily labeled zone at the area 17/18a border approximately 9% of all of the cells in layer II/III are callosally projecting nonpyramidal cells, and 70% of them are callosally projecting pyramidal cells. Light and electron microscopic examinations indicate that the nonpyramidal neurons are a heterogeneous group which consists of small multipolar neurons, large multipolar neurons, small bipolar neurons, and large bipolar neurons. To investigate the ultrastructural appearance of the punctate HRP labeling, selected neurons have been examined in thin sections. In the electron microscope, the tetramethylbenzidine (TMB) reaction product appears as electron-dense crystals, while the diaminobenzidine (DAB) reaction product appears as dark, electron-dense material which fills the lysosomes. These lysosomes occasionally have a halo of reaction product, but often they are not morphologically distinguishable from dark lysosomes present within neurons from control animals in which the darkening results from staining the thin sections with lead citrate and uranyl acetate. However, labeled neurons possess more dark lysosomes than neurons from control animals. These additional dark lysosomes presumably contain the HRP reaction product visible by light microscopy.     

Dark cells of cystinosis: occurrence in renal allografts

Twenty-four biopsies of renal allografts, generally cadaveric, from 20 patients with cystinosis were examined by light, polarization, phase contrast, and electron microscopy. The unusual dark cells previously reported in the native kidneys and livers of patients with cystinosis were observed in 12 of the 24 biopsies. The cells were present in the interstitium in all of these 12 biopsies, in glomeruli in one biopsy, and in the tubular lumen in two biopsies. They were evident by light and electron microscopy in stained and unstained ultrathin sections, and could be discerned solely in Epon sections. The dark appearance resulted from the presence of dark, fine granular material in the cytoplasm and nucleus and in cytoplasmic inclusions. The cells were judged to be macrophages. They were present as early as 3 months following transplantation and bore no relationship to interstitial crystals or inflammation. The dark cells have two important implications: cystine storage may not be limited to lysosomes, and dark cells are a morphologic alternative to the traditional identifying configuration of cystine in tissues, namely crystals.     

Plastic casts and confocal laser scanning microscopy applied to the observation of enamel tubules in the red Kangaroo (Macropus rufus)

Scanning electron microscopy for plastic casts and confocal laser scanning microscopy for Villanueva bone-stained ground sections were used together to observe enamel tubules in red kangaroo molars. Although the tubular structures such as terminals, bends, expansions, splits, divergences and rejoinings in this species were within the variations of marsupial species, their morphological characteristics were demonstrated with extremely clear and persuasive images. Thus, the combined observations of plastic casts by scanning electron microscopy and Villanueva bone-stain sections by confocal laser scanning microscopy were found to be of value for the investigation of enamel tubules and tubular structures in other hard tissues.     

Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout,and first description of erythrophore ultrastructure in salmonids

Skin pigmentation in animals is an important trait with many functions. The present study focused on two closely related salmonid species, marble trout (Salmo marmoratus) and brown trout (S. trutta), which display an uncommon labyrinthine (marble‐like) and spot skin pattern, respectively. To determine the role of chromatophore type in the different formation of skin pigment patterns in the two species, the distribution and ultrastructure of chromatophores was examined with light microscopy and transmission electron microscopy. The presence of three types of chromatophores in trout skin was confirmed: melanophores; xanthophores; and iridophores. In addition, using correlative microscopy, erythrophore ultrastructure in salmonids was described for the first time. Two types of erythrophores are distinguished, both located exclusively in the skin of brown trout: type 1 in black spot skin sections similar to xanthophores; and type 2 with a unique ultrastructure, located only in red spot skin sections. Morphologically, the difference between the light and dark pigmentation of trout skin depends primarily on the position and density of melanophores, in the dark region covering other chromatophores, and in the light region with the iridophores and xanthophores usually exposed. With larger amounts of melanophores, absence of xanthophores and presence of erythrophores type 1 and type L iridophores in the black spot compared with the light regions and the presence of erythrophores type 2 in the red spot, a higher level of pigment cell organisation in the skin of brown trout compared with that of marble trout was demonstrated. Even though the skin regions with chromatophores were well defined, not all the chromatophores were in direct contact, either homophilically or heterophilically, with each other. In addition to short‐range interactions, an important role of the cellular environment and long‐range interactions between chromatophores in promoting adult pigment pattern formation of trout are proposed.     

Differences in the chemical expression of rat primary afferent neurons which innervate skin, muscle or joint

The fluorescent dye Fast Blue was injected in anaesthetized rats into either skin, muscle or knee joint of the hindlimb. Following retrograde transport of the dye to lumbar dorsal root ganglia, the cell bodies of primary afferent neurons innervating these different target tissues were identified in ganglion sections by fluorescence microscopy. The sections were processed to demonstrate activity of the enzyme thiamine monophosphatase, or immunoreactivity to calcitonin gene-related peptide, substance P, or somatostatin, in Fast Blue labelled neurons. In all cases immunoreactivity to the antineurofilament antibody RT97 was used to classify dorsal root ganglion cells as being either small dark (RT97 negative, unmyelinated axons) or large light (RT97 positive, myelinated axons). The proportion of small dark cells labelled from each target decreased in the order: skin, muscle, joint. Thiamine monophosphatase and somatostatin were present only in small dark cells, while calcitonin gene-related peptide and substance P were found in both small dark and large light cells. In large light cells of all three targets, more contained calcitonin gene-related peptide than substance P. Among small dark cells, thiamine monophosphatase and somatostatin were found predominantly in skin afferents, while calcitonin gene-related peptide and substance P were more common in muscle and joint afferents. The chemical expression of primary afferents is therefore characteristic of the peripheral target they innervate. This could reflect either a maintained influence of the target on the afferents, or the factors which operate only during development.     

The spatial distribution of focal stacks within the inner enamel layer of mandibular mouse incisors

Focal stacks are an alternative spatial arrangement of enamel rods within the inner enamel of mandibular mouse incisors where short rows comprised of 2–45 enamel rods are nestled at the side of much longer rows, both sharing the same rod tilt directed mesially or laterally. The significance of focal stacks to enamel function is unknown, but their high frequency in transverse sections (30% of all rows) suggests that they serve some purpose beyond representing an oddity of enamel development. In this study, we characterized the spatial distribution of focal stacks in random transverse sections relative to different regions of the inner enamel and to different locations across enamel thickness. The curving dentinoenamel junction (DEJ) in transverse sections complicated spatial distribution analyses, and a technique was developed to “unbend” the curving DEJ allowing for more linear quantitative analyses to be carried out. The data indicated that on average there were 36 ± 7 focal stacks located variably within the inner enamel in any given transverse section. Consistent with area distributions, focal stacks were four times more frequent in the lateral region (53%) and twice as frequent in the mesial region (33%) compared to the central region (14%). Focal stacks were equally split by tilt (52% mesial vs. 48% lateral, not significant), but those having a mesial tilt were more frequently encountered in the lateral and central regions (2:1) and those having a lateral tilt were more numerous in the mesial region (1:3). Focal stacks having a mesial tilt were longer on average compared to those having a lateral tilt (7.5 ± 5.6 vs. 5.9 ± 4.0 rods per row, p < 0.01). There was no relationship between the length of a focal stack and its location within the inner enamel. All results were consistent with the notion that focal stacks travel from the DEJ to the outer enamel the same as the longer and decussating companion rows to which they are paired. The spatial distribution of focal stacks within the inner enamel was not spatially random but best fit a null model based on a heterogenous Poisson point process dependent on regional location within the transverse plane of the enamel layer.     

Characteristics of the transverse 2D uniserial arrangement of rows of decussating enamel rods in the inner enamel layer of mouse mandibular incisors

The 2D arrangement of rows of enamel rods with alternating (decussating) tilt angles across the thickness of the inner layer in rat and mouse incisor enamel is well known and assumed to occur in a uniform and repetitive pattern. Some irregularities in the arrangement of rows have been reported, but no detailed investigation of row structure across the entire inner enamel layer currently exists. This investigation was undertaken to determine if the global row pattern in mouse mandibular incisor enamel is predominately regular in nature with only occasional anomalies or if rows of enamel rods have more spatial complexity than previously suspected. The data from this investigation indicate that rows of enamel rods are highly variable in length and have complex transverse arrangements across the width and thickness of the inner enamel layer. The majority of rows are short or medium in length, with 87% having < 100 rods per row. The remaining 13% are long rows (with 100–233 rods per row) that contain 46% of all enamel rods seen in transverse sections. Variable numbers of rows were associated with the lateral, central and mesial regions of the enamel layer. Each region contained different ratios of short, medium and long rows. A variety of relationships was found along the transverse length of rows in each region, including uniform associations of alternating rod tilts between neighboring rows, and instances where two rows having the same rod tilt were paired for variable distances then moved apart to accommodate rows of opposite tilt. Sometimes a row appeared to branch into two rows with the same tilt, or conversely where two rows merged into one row depending upon the mesial‐to‐lateral direction in which the row was viewed. Some rows showed both pairing and branching/merging along their length. These tended to be among the longest rows identified, and they often crossed the central region with extensions into the lateral and mesial regions. The most frequent row arrangement was a row of petite length nestled at the side of another row having the same rod tilt (30% of all rows). These were termed ‘focal stacks’ and may relate to the evolution of uniserial rat and mouse incisor enamel from a multilayered ancestor. The mesial and lateral endpoints of rows also showed complex arrangements with the dentinoenamel junction (DEJ), the inner enamel layer itself, and the boundary area to the outer enamel layer. It was concluded that the diversity in row lengths and various spatial arrangements both within and between rows across the transverse plane provides a method to interlock the enamel layer across each region and keep the enamel layer compact relative to the curving DEJ surface. The uniserial pattern for rows in mouse mandibular incisors is not uniform, but diverse and very complex.     

The effect of strontium,cobalt and fluoride on rat incisor enamel formation

This investigation examined ultrastructurally the entire period of development of alterations in formative ameloblasts and the enamel which they produce following injection with fluoride, strontium, and cobalt ions. Rats injected with these ions were sacrificed at intervals of 1, 2, 4, 8, 16, 24 and 48 hours to elucidate the sequence and detail of cytologic and cell product alterations which occur. Undecalcified sections of rat incisor teeth were studied using electron microscopy and microradiography. All three ions initially produced disturbances in cell morphology and enamel formation consisting of dark globules, vacuoles, and pooling of stippled material on the enamel surface. While a period of decreased crystal formation occurred after injection with all three ions, only cobalt responses included a period of apparently complete absence of crystal formation. The hypermineralized layers occurring in the altered enamel are attributed to changes in the rate of enamel matrix formation and duration of its exposure to tissue fluids. Morphologic changes in Tomes' process were observed at the time of formation of abnormal enamel following injection of all three ions. These observations are compared with previous studies of altered enamel formation and analyzed with the goal of learning more about the mechanisms of amelogenesis.     

The morphologic relationship of light and dark cells of the collecting tubule in potassium-depleted rats.

The luminal surface of collecting tubule cells in the inner stripe of the renal medulla in normal and potassium-depleted rats was studied by scanning electron microscopy. In normal rats the luminal surfaces were of two types. One cell type was sparsely covered with small bulbous microvilli and had either a single or double cilium. This type corresponds to the light cell seen in transmission electron microscopy. The second cell type was covered by prominent microplicae and represents the dark cell observed in transmission electron microscopy. In potassium-depleted animals, numerous cells with a morphologic appearance of intermediate forms were identified. By scanning electron microscopy, the luminal surface of these cells was covered by a mixed population of villi and microplicae in different stages of development and often showed cilia, which were previously considered to exist only on light cells. On the basis of these morphologic findings, we conclude that the dark and light cells are not different cell types but rather represent different forms of a single type of cell.     

The effect of strontium, cobalt and fluoride on rat incisor enamel formation.

This investigation examined ultrastructurally the entire period of development of alterations in formative ameloblasts and the enamel which they produce following injection with fluoride, strontium, and cobalt ions. Rats injected with these ions were sacrificed at intervals of 1, 2, 4, 8, 16, 24 and 48 hours to elucidate the sequence and detail of cytologic and cell product alterations which occur. Undecalcified sections of rat incisor teeth were studied using electron microscopy and microradiography. All three ions initially produced disturbances in cell morphology and enamel formation consisting of dark globules, vacuoles, and pooling of stippled material on the enamel surface. While a period of decreased crystal formation occurred after injection with all three ions, only cobalt responses included a period of apparently complete absence of crystal formation. The hypermineralized layers occurring in the altered enamel are attributed to changes in the rate of enamel matrix formation and duration of its exposure to tissue fluids. Morphologic changes in Tomes' process were observed at the time of formation of abnormel following injection of all three ions. These observations are compared with previous studies of altered enamel formation and analyzed with the goal of learning more about the mechanisms of amelogenesis.     

Birefringence of the Secretory-Stage Enamel Organic Extracellular Matrix from Rats Submitted to Successive Injections of Bisphosphonates

The aim of the present study was to assess birefringence of the secretory-stage enamel organic extracellular matrix (ECM) and mechanical properties of mature enamel from rats treated with bisphosphonates. Longitudinal sections were obtained from upper incisors of rats that had been submitted to injections of bisodic etidronate (8 mg/Kg/day), sodium alendronate (30 μg/Kg/day), or sodium chloride as control (8 mg/Kg/day) for 42 days. Sections were immersed in 80% glycerin for 30 min and optical retardation of birefringence brightness in the secretory-stage enamel organic ECM was determined in nanometers. Etidronate-treated rats exhibited extensive morphological changes in the secretory-stage enamel organic ECM inclusive nonbirefringent conspicuous incremental lines, but presented optical retardation values similar to those showed by control rats (p > 0.05). Birefringence of secretory enamel organic ECM from etidronate rats presented an irregular aspect. Alendronate-treated rats did not show morphological alterations in the secretory-stage enamel organic ECM, however, they presented significant reduction in optical retardation of birefringence brightness when compared with control and etidronate rats (p < 0.01). Alendronate and etidronate groups exhibited reductions of ≈6–10% in mature enamel cross-sectional microhardness when compared with control group (p < 0.01). Scanning electron microscopy analysis showed extensive alterations in mature enamel only from etidronate group with absence of enamel rods. The present work shows that bisphosphonates can affect the birefringence of the secretory-stage enamel organic ECM. The results presented here suggest that alterations in the supramolecular organization of the secretory-stage enamel organic ECM are a plausible mechanism by which environmental factors may cause enamel defects.