Confocal microscopy of dentinal tubules in human tooth stained with alizarin red

 The present study was designed to analyze the structures of dentinal tubules by confocal microscopy. Undecalcified ground sections of human teeth were stained with alizarin red in 0.1% KOH aqueous solution, and examined by confocal microscopy. Alizarin red stained dentinal tubules, interglobular dentine, granular layer of Tomes, and the surface of dentine. Interglobular dentine was seen between the outer and middle layers of coronal dentine. At the outer layer of coronal dentine, the dentinal tubules were thin and showed numerous branches. At the middle layer of coronal dentine, dentinal tubules displayed two types. The type I tubules are the dentinal tubules that do not show any nodular structures and the type II tubules are the dentinal tubules that appear bamboo-like with many nodules. In the cross section through the type II tubules, the nodules appeared as fine circular tubules surrounding the dentinal tubules. The circular tubules of nodules adhered to one side of the dentinal tubules. When the fluorescence images were compared with the images taken by transmission light mode, the fluorescence of dentinal tubules was seen at the inner surface of dentinal tubules, and the fluorescence of nodules was seen at interface between peritubular and intertubular dentine. Most of the dentinal tubules were of the type II tubules in the teeth from older individuals, whereas the type II tubules were scarce in the teeth from younger individuals. At the inner layer of coronal dentine, the dentinal tubules have no nodules and branches were scarce. The dentinal tubules of radicular dentine were different from those of coronal dentine. Most of the dentinal tubules were the type I tubules. Numerous fine branches were seen at the outer and middle layers of radicular dentine. No interglobular dentine was seen in the root except at the cervical part, and the granular layer of Tomes was also positive with alizarin red. At the cervical part of the root, interglobular dentine was present and the dentinal tubules displayed types I and II. Accepted: 19 August 1998     

The dentinal structure of equine incisors: a light and scanning electron-microscopic study

This paper gives an anatomical overview of the dentinal structure in equine incisor teeth with special reference to the three-dimensional organization, the number and the diameter of the dentinal tubules. The spatial arrangement of equine dentine was examined by scanning electron microscopy of occlusal surfaces and longitudinally fractured teeth and by light microscopy of both decalcified and ground sections. The dentinal tubules of the peripherally situated primary dentine were directly continuous with those of the circumpulpal secondary dentine. The tubules had numerous side branches along their entire course and ramified into terminal branches near the dentino-enamel junction. Tubules of tertiary dentine, situated in the centre of the dental star, were few in number and not continuous with those of the surrounding secondary dentine. On non-etched incisors tubular orifices were clogged by an amorphous smear layer that covered the occlusal surface. On etched occlusal surfaces the numerical tubular density was calculated in different zones. The largest number of tubules per unit area was situated in the secondary dentine forming the dark periphery of the dental star, whereas the smallest number was present in the tertiary dentine located in the pale centre of the dental star. Dentinal tubular diameters were measured at various distances from the pulp. They were widest near the pulpal wall and narrowed progressively towards the dentino-enamel junction. Tubular widths measured on etched occlusal surfaces were considerably larger than those measured on undecalcified incisors due to the removal of intratubular dentine during the etching process.     

Development of coronal cementum in hypsodont horse cheek teeth

The horse is a grazing herbivore whose cheek teeth are hypsodon; that is, they possess long crowns that are completely covered by coronal cement at eruption. For elucidation of the sequential events in the formation of this coronal cementum in the mandibular horse cheek teeth, in the present study the lower 3rd permanent premolar teeth (PM₄) from 3.5‐, 4‐, and 5‐year‐old horses were compared by using radiography, microcomputed tomography (Miro‐CT), light microscopy (LM), and scanning electron microscopy (SEM). The present study clearly showed that prior to coronal cementogenesis tartrate‐resistant acid phosphatase (TRAP)‐positive odontoclasts resorbed on the enamel surface of the reserve crown in horse cheek tooth. Enamel resorption areas were relatively narrow, and started from the cuspal tips, and moved in the apical direction during tooth development. A primary cementum was initially deposited on the irregularly pitted enamel–cementum junction (ECJ) of the infolding and peripheral enamel. The infolding cementum filled grooves completely by the time of tooth eruption. On the other hand, in the peripheral cementum, the secondary and tertiary cementum layers were sequentially deposited on the primary cementum. These two cementum layers were sites for the insertion of the periodontal ligaments, and were continually laid down on the primary cementum coronally rather than apically throughout the life. The results of the present study suggest that the coronal cementum of horse cheek teeth is a multistructural and multifunctional tissue, meeting the requirements of its many different functions. Anat Rec, 297:716–730, 2014. © 2014 Wiley Periodicals, Inc.     

Mineral Apposition Rates in Coronal Dentine of Mandibular First Molars in Soay Sheep: Results of a Fluorochrome Labeling Study

We studied the spatio‐temporal variation of mineral apposition rate (MAR) in postnatally formed coronal dentine of mandibular first molars from Soay sheep repeatedly injected with different fluorochromes. MAR declined along the cuspal to cervical crown axis, and from early to late formed dentine, that is, from the dentine at the enamel‐dentine‐junction (EDJ) to the dentine adjacent to the dentine‐pulp‐interface (DPI). Highest mean MARs (about 21 µm/day) were recorded in cuspal dentine formed in the period of 28‐42 days after birth. Lowest values (<2 µm/day) were recorded in late‐formed (secondary) dentine close to the DPI. The high MARs recorded in the dentine of the cuspal crown portions enable the formation of a large tooth crown within a relatively short period of less than one year. The established MARs in the dentine of the different crown portions of sheep molars will allow a precise determination of the timing of stress events affecting dentine formation. They are also helpful for devising sampling protocols in studies of trace element or stable isotope distributions in sheep dentine aimed at assessing temporal variation of incorporation into forming dentine. Such data are useful in a variety of contexts, including, for example, the exposure to pollutants and the reconstruction of husbandry practices or feeding regimes. Anat Rec, 301:902–912, 2018. © 2017 Wiley Periodicals, Inc.     

Changes in the structure of dentine from cheek teeth of deer chronically exposed to high levels of environmental fluoride

The purpose of this study was to examine, using backscattered electron imaging, the changes occurring in the structure of the dentine including giant tubules in the teeth of deer chronically exposed to fluoride. The primary and secondary dentine was characterised by the presence of interglobular dentine and regular bands of hypo- and hypermineralised dentine. Giant tubules in the unworn teeth of animals exposed to low and high levels of fluoride were characterised by a large lumen and hypermineralised rim in which there were calcospherites. With the occlusal wear of the teeth tubules beneath the immediate surface of the exposed dentine in teeth from fluorotic animals became occluded with mineral, but in low fluoride animals some tubules at the surface were not occluded with mineral. It is suggested that mineral which occludes the tubules may come from both intrinsic and extrinsic sources. The presence of some open tubules at the immediate surface in the worn teeth of animals exposed to low fluoride suggests an extrinsic source for this mineral.     

In situ analysis of gelatinolytic activity in human dentin

Matrix metalloproteinases (MMPs) such as gelatinases are differentially expressed in human tissues. These enzymes cleave specific substrates involved in cell signaling, tissue development and remodeling and tissue breakdown. Recent evidences show that gelatinases are crucial for normal dentin development and their activity is maintained throughout the entire tooth function in the oral cavity. Due to the lack of information about the exact location and activity of gelatinases in mature human dentin, the present study was designed to examine gelatinolytic levels in sound dentin. In situ zymography using confocal microscopy was performed on both mineralized and demineralized dentin samples. Sites presenting gelatinase activity were identified throughout the entire biological tissue pursuing different gelatinolytic levels for distinct areas: predentin and dentinal tubule regions presented higher gelatinolytic activity compared to intertubular dentin. Dentin regions with higher gelatinolytic activity immunohistochemically were partially correlated with MMP-2 expression. The maintenance of gelatinolytic activity in mature dentin may have biological implications related to biomineralization of predentin and tubular/peritubular dentinal regions, as well as regulation of defensive mechanisms of the dentin-pulp complex.     

Distribution of interglobular dentine in human tooth roots

The present study was designed to examine the distribution of interglobular dentine in human tooth roots. The material comprised 17 teeth, of which 3 were premolars extracted for orthodontic reasons from children 10-12 years of age and the other teeth (4 incisors, 3 canines and 7 molars) were extracted for periodontitis from individuals aged 32-63 years. All teeth were free of caries and cervical dentine defects. Ground sections of the teeth cut longitudinally were stained with basic fuchsin and observed by fluorescence and confocal microscopy as well as transmitted light microscopy. Basic fuchsin stained the dentinal tubules, interglobular dentine and the granular layer of Tomes. These structures appeared intense blue to faint violet with transmitted light microscopy, whereas their staining displayed intense fluorescence with fluorescence microscopy. Therefore, the interglobular dentine could be detected more sensitively with fluorescence and confocal microscopy than with transmitted light microscopy. Typical interglobular dentine was present in coronal dentine in most of the teeth. In the radicular dentin, position and size of the interglobular dentine was different among the teeth examined. Most of the teeth had the interglobular dentine in the cervical part of the roots (type A). Two premolars displayed the interglobular dentine in the coronal half of the root (type B). The types A and B contained large interglobular areas. A small amount of interglobular dentine was restricted to the apical half of the roots of two canines and one molar (type C). In contrast to types A and B which were seen at both labial or buccal and lingual sides of roots, the interglobular dentine of type C was seen only at one side, labial or lingual. Some of the tooth roots did not show any interglobular dentine (type D). Most of the incisors, canines and premolar were types A, B, and C, respectively, and the molars were mixed types A, C, and D. These results suggest that the factors affecting dentinogenesis during root formation are unique for each tooth.     

牙本质微结构性质的重新评价

目的相比以前一些模型,蛋白质-矿物质多孔力学模型被验证可以提供更精确的生物材料性质评价。因此,本文对牙本质微结构相互作用对其材料性质的影响进行了重新评价。方法采用蛋白质-矿物质多孔力学模型,讨论了牙小管、管周基质和管间基质相互作用对牙本质微结构性质的影响。结果通过分析发现,牙本质微结构力学性质依赖于牙小管的方向和牙小管中的压力值;同时,牙本质微结构力学性质会随着年龄以及管周基质与管间基质的蛋白质和矿物质分布而改变。结论文章中的理论分析和讨论说明牙小管、管周基质和管间基质相互作用对牙本质微结构性质有一定的影响,说明蛋白质-矿物质多孔力学模型的有效性,以及不考虑相互作用的微结构力学模型具有一定的局限性。     

Structure of the upper teeth of the filefish, Stephanolepis cirrhifer

The teeth of the filefish were investigated with light and scanning electron microscopy. Anterior and posterior rows of pleurodont teeth are present in the maxilla. The teeth in the anterior row possess a large labial and small lingual surface and the teeth in the posterior row possess a small labial and large lingual surface. The enameloid consists of three layers-the outer, middle, and inner layer. The outer layer consists of fine, parallel crystallites oriented perpendicular to the surface. They are similar to the crystallites in enamel derived from ectoderm. In the middle layer the crystallites form parallel bundles that are oriented in the same direction as that of the crystallites in the outer layer. In the inner layer bundles of crystallites interlace with each other. The tubular structures and the terminal branches of the dentinal tubules are located among the crystallites in the inner layer. In the dentine numerous dentinal tubules radiate from the pulp cavity toward the periphery and are seen to curve slightly in an S-shaped course. At the occlusal tip the alternating parabolic layers are seen and these are considered to be mineralized lines. The openings of the dentinal tubules are round or oval, surrounded by interlacing fibrils. The teeth are tightly fixed to the jawbone by bundles of fibrils. At the lower part of the lingual surface of the teeth in the anterior row and the labial surface of the teeth in the posterior row the bundles of fibrils start at the dentine and some fibrils run through connective tissue, while others terminate in projections of the jawbones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conservation of a 31-bp bovine subrepeat in centromeric satellite DNA monomers ofCervus elaphus and other cervid species

A centromeric satellite DNA clone was isolated from the genome of the European red deer (Cervus elaphus hippelaphus) and designated Ce-Pst1. This clone was localized to the centromeric region of all red deer chromosomes with the exception of a single pair of metacentric autosomes and the Y chromosome. DNA sequence analysis of the 806-bp Ce-Pst1 clone showed 73.0–78.9% sequence homology to four previously isolated cervid centromeric satellite DNA clones, suggesting that the Ce-Pst1 clone is yet another member of the major cervid centromeric satellite DNA family. Using a DNA sequence comparison system, internal 31-bp tandem subrepeats were found in the Ce-Pst1 clone as well as in the other previously reported cervid centromeric satellite DNA monomer sequences. A 31-bp consensus sequence was constructed for each cervid monomer clone and shown to be highly homologous to the 31-bp subrepeat consensus sequence found in bovine 1.715 centromeric satellite DNA. The identification of internal subrepeats in the satellite monomers studied could suggest that amplification of an ancestral 31-bp DNA sequence may have contributed to the genesis of major cervid centromeric satellite DNA. The homology between the 31-bp subrepeats found in cervid and bovid centromeric satellite DNAs substantiates the theory that amplification of this 31-bp DNA sequence may have occurred before the evolutionary separation of these two families 20–25 million years ago.accepted for publication by H. C. Macgregor     

Scanning microscopy of platypus teeth

Summary Anorganic unerupted developing teeth and airdired erupted teeth of the platypus (Ornithorhynchus anatinus) were examined in a scanning electron microscope and in a tandem scanning reflected light microscope. Typically mammalian developing fronts of enamel and dentine were identified in the anorganic unerupted specimens. The developing teeth were particularly small and fragile and the enamel elusive and difficult to examine in the normal way for morphological detail. Prepared fractured surfaces of unerupted specimens revealed preferentially oriented crystallite groups in the enamel generally perpendicular to the developing front and a highly globular, mineralized pattern in the dentine with fine diameter, sparsely distributed dentinal tubules.Although optically homogeneous, the enamel of both developing and mature teeth displayed well-defined incremental lines, radial clefts, crystallite domains of variable size and outline, and fine tubules when examined by high contrast, back-scattered electron imaging. The enamel is prismatic only in part; well-formed, regular prisms not being a primary feature of platypus enamel. This can be related to the variability inherent in the developing surface and the thinness of the enamel layer. No surface was found which could be confidently identified as cementum; those developing surfaces not covered by enamel displaying small calcospherites which elsewhere marked the outer aspect of the dentine.     

Citric acid etching of cervical sclerotic dentin lesions: an AFM study

Atomic force microscopy (AFM) has been used to determine microstructural changes, etching rates of peritubular dentin, and intertubular dentin recession during demineralization in dilute acidic solutions. These studies have not included many forms of altered dentin, including noncarious sclerotic root dentin associated with Cl V (abfraction) lesions. We sought to determine microstructural changes and recession rates during demineralization in citric acid (pH 2.5, 0.018M) for the transparent/sclerotic zone. Highly polished dentin disks were prepared from teeth with noncarious C1 V lesions (n = 3) and compared with normal root dentin (n = 3). Samples were etched at 5-s intervals for 1 min and at longer intervals up to 30 min. The depth changes in various portions of the dentin with respect to the reference layer were measured and changes in microstructure observed in solution in the wet cell of the AFM. In sclerotic dentin, most tubule lumens were occluded with crystalline deposits that etched more slowly than the other dentin components, but etching rates could not be determined due to their irregular geometry. The intertubular dentin recession quickly reached a plateau after a depth change of <1 microm for all dentin types, in agreement with prior work. Mixed linear regression models indicated an important difference between the etching of sclerotic intertubular dentin and that of non-sclerotic root dentin that became apparent after 600 s (p = 0.037). The sclerotic intertubular dentin underwent less depth change at the plateau (558 nm) compared to normal root dentin (744 nm). In addition, normal root dentin underwent significantly greater recession than coronal dentin (p = 0.002). The results of this study indicate that intertubular sclerotic dentin from Cl V lesions etches differently than normal root dentin, and this may help explain the difficulties in restoring such lesions with current bonding procedures.     

Extensive and coincident side branching of dentinal tubules

Slices of decalcified cat's canine teeth were impregnated with silver nitrate. The stain was taken up by the dentinal tubules. In sections 100 microns thick cut parallel to the long axis of the tooth, dark staining lines were seen running at right angles to the dentinal tubules. Examination at higher power and in thinner sections showed this appearance to be due to the coincident side branching of dentinal tubules. The side branches anastomosed and formed a series of lateral channels in the dentine. These channels may allow for the lateral spread of dentinal caries and of stimuli applied to the peripheral dentine.     

Evolutionary trajectories of tooth histology patterns in modern sharks (Chondrichthyes,Elasmobranchii)

During their evolutionary history, modern sharks developed different tooth mineralization patterns that resulted in very distinct histological patterns of the tooth crown (histotypes). To date, three different tooth histotypes have been distinguished: (i) orthodont teeth, which have a central hollow pulp cavity in the crown, encapsulated by a prominent layer of dentine (orthodentine); (ii) pseudoosteodont teeth, which have their pulp cavities secondarily replaced by a dentinal core of porous dentine (osteodentine), encased by orthodentine; and (iii) osteodont teeth, which lack orthodentine and the whole tooth crown of which consists of osteodentine. The aim of the present study was to trace evolutionary trends of tooth mineralization patterns in modern sharks and to find evidence for the presence of phylogenetic or functional signals. High resolution micro-computed tomography images were generated for the teeth of members of all nine extant shark orders and the putative stem group †Synechodontiformes, represented here by three taxa, to examine the tooth histology non-destructively. Pseudoosteodonty is the predominant state among modern sharks and represents unambiguously the plesiomorphic condition. Orthodonty evolved several times independently in modern sharks, while the osteodont tooth histotype is only developed in lamniform sharks. The two shark orders Heterodontiformes and Pristiophoriformes showed highly modified tooth histologies, with Pristiophorus exhibiting a histology only known from batomorphs (i.e. rays and skates), and Heterodontus showing a histological difference between anterior and posterior teeth, indicating a link between its tooth morphology, histology and durophagous lifestyle. The tooth histotype concept has proven to be a useful tool to reflect links between histology, function and its taxonomic value for distinct taxa; however, a high degree of variation, especially in the pseudoosteodont tooth histotype, demonstrates that the current histotype concept is too simplistic to fully resolve these relationships. The vascularization pattern of the dentine might offer new future research pathways for better understanding functional and phylogenetic signals in the tooth histology of modern sharks.     

An investigation of pulp capillaries and tight junctions between odontoblasts in cats

Summary The relative roles of capillaries and odontoblasts in the process of dentinogenesis and in pulp reactions to trauma and pathology are not clear. Contributing to the problem is the paucity of information on odontoblast —capillary relationships and tight junctions between odontoblasts. Using light microscopy the capillaries have now been examined in semithin transverse sections of perfusion fixed teeth at different positions in the long axis from the apical foramina to the pulp horns. Odontoblastic capillaries were prominent in the coronal and middle regions of canines and present at the same levels of incisors. In the pulp horns and just coronal to the pulp horns capillaries were all subodontoblastic but near the apex there were also a few odontoblastic capillary profiles. Transmission electron microscopy on ultrathin sections revealed that a high proportion of middle and coronal odontoblastic capillary profiles were fenestrated but subodontoblastic profiles coronal to the pulp horns were the most fenestrated. In a search for tight junctions in ultrathin sections some typical strands were observed between odontoblasts. The difficult of obtaining the latter evidence was explained by the cellular arrangement of the odontoblasts which differed markedly from an ideal parallel, apically coplanar arrangement. The results question the possibility that there is a direct exchange of materials between pulp capillaries and dentine in teeth of limited growth and provide a baseline for future experiments to test the permeability of the odontoblast layer. Present address: Oral Biology Department, Northwestern University Dental School, 303E Chicago Avenue, Chigaco, IL 60611, USA     

A combined scanning electron microscopy and immunofluorescence study demonstrating that the odontoblast process extends to the dentinoenamel junction in human teeth

The extent of the odontoblast cell process has been the subject of controversy for many years. Using SEM we have examined the extent and morphology of the process on dentine surfaces of human teeth which were partially demineralized and collagenase digested. Third molars were extracted and split; the dentine surface was demineralized, digested by bacterial collagenase, fixed with glutaraldehyde, postfixed in osmium tetroxide, and prepared for SEM investigation. The SEM study revealed the presence of many processlike structures which extended from the odontoblast cell bodies up to the dentinoenamel junction (DEJ). These processes demonstrated lateral and terminal branching and some of them terminated in distended spheres. We have also applied an immunofluorescence technique at the light microscope level to these exposed dentinal surfaces to localize the intracellular microtubules. For this, a second series of third molars was processed in the same manner as for the SEM up to the fixation stage. Teeth were then fixed in periodate-lysine-paraformaldehyde, postfixed in -20 degrees C acetone, and then incubated with affinity-purified rabbit antitubulin antibodies, followed by fluorescein-conjugated goat antirabbit IgGs. Intratubular immunofluorescence labelling for tubulin was evident from the odontoblast cell bodies up to the DEJ. The presence of the tubulin-containing structures extending to the DEJ supports the hypothesis that the structures observed with the SEM are odontoblast processes and that the odontoblast processes do extend to the DEJ.     

The blood vascular architecture of the rat testis: a scanning electron microscopic study of corrosion casts followed by light microscopy of tissue sections

The blood vascular bed of the rat testis was studied by scanning electron microscopy (SEM) of corrosion casts and by light microscopy of tissue sections. The testicular artery penetrates the pampiniform plexus and gives rise to the intertubular arterioles. Each of these arterioles courses in the intertubular connective tissue column, and gives off intertubular and peritubular capillaries. The intertubular capillaries pass the intertubular connective tissue column, whereas the peritubular capillaries reach the peritubular connective tissue sheet. The intertubular and peritubular capillaries anastomose with each other and converge into the intertubular venules in the intertubular connective tissue columns. Thus, the blood vascular bed of the rat testis consists of hexago- or pentago-columnar capillary networks which commonly surround the seminiferous tubules. The Leidig's cells are preferentially observed in the intertubular connective tissue columns. One of the intertubular capillaries is consistently thick, and directly continues into the intertubular venules (arteriolo-venular capillary channels), which finally drain into the pampiniform plexus. These findings suggest that the male sex hormone, testosterone, as secreted by the Leidig's cells, is discharged into the intertubular capillaries and then mainly carried by the arteriolo-venular capillary channels and intertubular venules into the pampiniform plexus. This specialized drainage may ensure the presence of highly concentrated testosterone in the pampiniform plexus and allow the testosterone-exchange from the pamipiniform plexus to the testicular artery. The arteriolo-venular capillary channels may also eliminate blood congestion in the testis to enhance the efficiency of the heat-exchange mechanism between the testicular artery and pampiniform plexus. Many arterio-arterial and arterio-venous anastomoses occur, which may regulate the blood flow within the testis.     

Scanning Electron Microscope Characterization of Erosive Enamel in Human Teeth

This study aimed to examine the surface characteristics of erosive enamel in extracted human teeth by scanning electron microscopy. Morphologic changes in naturally eroded enamel depend on the stages of dental erosion. In its early stages, the enamel surfaces show a honeycomb appearance due to the dissolution of enamel rod ends. In its advanced stages, the erosive process involves the underlying dentin and the eroded dentin shows exposed dentinal tubules and the dentinal matrix may be exposed due to the dissolution of the peri- and intertubular dentin. Evidence of remineralization is seen at the early stage of natural dental erosion.     

Digital Image Analysis in Dental Research Applied for Treatment of Fissures on Occlusal Surfaces of Premolars

The aim of this paper was to quantitatively assess caries changes of teeth by using digital image analysis. Digital images of stained sections of crowns of teeth were acquired with a computer-assisted light microscope. In each image, spots representing the main and total demineralization of enamel were segmented to determine their area. The area of total demineralization was significantly different between premolars with sealed fissures and unprotected premolars as indicated by the Mann–Whitney test. Fissures on occlusal surfaces of premolars were characterized by their width, height, and distance from the bottom of the fissure to the enamel–dentin junction. This distance was also significantly different between protected and unprotected teeth. The results of our in vitro studies of enamel lesions allow us to plan an effective diagnosis, prophylaxis, and treatment of early caries changes in in vivo conditions. © 2003 Biomedical Engineering Society. PAC2003: 8764Rr, 8757Ra, 8757Nk, 8780Pa     

The Control of Ingress of Albumin into Developing Enamel from Adjacent Dentine of the Rat Incisor

The present study used an immunohistochemical approach to map the distribution of albumin within the dentine adjacent to the developing enamel of both impeded and unimpeded rat incisors to determine if the dentine could be a possible route of entry for this protein into the developing enamel matrix. In dentine adjacent to the secretory and transition stages of the developing enamel, the dentinal tubules were labelled only over approximately the pulpal quarter of their length. The bulk of the dentine showed no labelling. However, labelling within the dentine appeared at the ADJ at a position approximately 2.5 mm occlusal to the distal root of the first molar, adjacent to enamel with no visible residual matrix. The results of this study suggest that adventitious ingress of albumin into enamel from the dentine is restricted during enamel secretion and can only potentially occur once enamel maturation has been initiated.