Atomic force microscopy of collagen structure in bone and dentine revealed by osteoclastic resorption

Mineralised tissues such as bone consist of two material phases: collagen protein fibrils, secreted by osteoblasts, form model structures for subsequent deposition of mineral, calcium hydroxyapatite. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair. Bone active drugs have recently been developed for skeletal diseases, and there is revived interest in changes in the structure of mineralised tissues seen in disease and upon treatment. The resolution of atomic force microscopy and use of unmodified samples has enabled us to image bone and dentine collagen exposed by the natural process of cellular dissolution of mineralised matrix. The morphology of bone and dentine has been analysed when fully mineralised and after osteoclast-mediated bone resorption, and compared with results from other microscopy techniques. Banded type I collagen, with 66.5+/-1.4 nm axial D-periodicity and 62.2+/-7.0 nm diameter, has been identified within resorption lacunae in bone and 69.4+/-4.3 nm axial D-periodicity and 140.6+/-12.4 nm diameter in dentine substrates formed by human and rabbit osteoclasts, respectively. This observation suggests a route by which the material and morphological properties of bone collagen can be analysed in situ, compared with collagen from non-skeletal sites, and contrasted in diseases of medical importance, such as osteoporosis, where skeletal tissue is mechanically weakened.     

First-generation teeth in nonmammalian lineages: Evidence for a conserved ancestral character?

The present study focuses on the main characteristics of first-generation teeth (i.e., the first teeth of the dentition to develop in a given position and to become functional) in representatives of the major lineages of nonmammalian vertebrates (chondrichthyans, actinopterygians, and sarcopterygians: dipnoans, urodeles, squamates, and crocodiles). Comparative investigations on the LM and TEM level reveal the existence of two major types of first-generation teeth. One type (generalized Type 1) is characterized by its small size, conical shape, atubular dentine, and small pulp cavity without capillaries and blood vessels. This type is found in actinopterygians, dipnoans, and urodeles and coincides with the occurrence of short embryonic periods in these species. The other type assembles a variety of first-generation teeth, which have in common that they represent miniature versions of adult teeth. They are generally larger than the first type, have more complex shapes, tubular dentine, and a large pulp cavity containing blood vessels. These teeth are found in chondrichtyans, squamates, and crocodiles, taxa which all share an extended embryonic period. The presence in certain taxa of a particular type of first-generation teeth is neither linked to their phylogenetic relationships nor to adult body size or tooth structure, but relates to the duration of embryonic development. Given that the plesiomorphic state in vertebrates is a short embryonic development, we consider the generalized Type 1 first-generation tooth to represent an ancestral character for gnathostomes. We hypothesize that an extended embryonic development leads to the suppression of tooth generations in the development of dentition. These may still be present in the form of rudimentary germs in the embryonic period. In our view, this generalized Type 1 first-generation teeth has been conserved through evolution because it represents a very economic and efficient way of building small and simple teeth adapted to larval life. The highly adapted adult dentition characteristic for each lineage has been possible only through polyphyodonty.     

Quantitative analyses of the biomineralization of different hard tissues

The primary crystallites of the different developing hard tissues have an apatite structure. However, they have crystal lattice distortions representing an intermediate state between amorphous and fully crystalline. We have applied energy-filtering transmission electron microscopy in the selected area electron diffraction mode to analyse different stages of crystal formation in dentine, bone, enamel and inorganic apatite mineral. We have obtained quantitative information on the degree of crystal lattice distortion using the paracrystal theory of Hosemann and Bagchi.
We have found that the early formed crystallites of the hard tissues being analysed have a paracrystalline character comparable to biopolymers. However, with maturation, the lattice fluctuations of the crystallites of the hard tissues bone, enamel and dentine decrease to form a typical (para)crystalline character. Also the decrease of the organic proportion in the matrix corresponds to the decrease of the lattice fluctuation of the crystallites in the different hard tissues during maturation.     

天然牙/纯钛摩擦学特性的研究

对人体天然牙齿与TA2钛球组成的摩擦副进行了试验研究,结合显微分析,研究了天然牙不同层面的摩擦学行为。结果表明牙冠外层的釉质具有优良的摩擦学特性,牙釉质/牙本质交界面的耐磨性稍逊于外层釉质,但明显高于牙本质。     

Prospects for tooth regeneration in the 21st century: a perspective

The prospects for tooth regeneration in the 21st century are compelling. Using the foundations of experimental embryology, developmental and molecular biology, the principles of biomimetics (the mimicking of biological processes), tooth regeneration is becoming a realistic possibility within the next few decades. The cellular, molecular, and developmental "rules" for tooth morphogenesis are rapidly being discovered. The knowledge gained from adult stem cell biology, especially associated with dentin, cartilage, and bone tissue regeneration, provides additional opportunities for eventual tooth organogenesis. The centuries of tooth development using xenotransplantation, allotransplantation, and autotransplantation have resulted in many important insights that can enhance tooth regeneration. In considering the future, several lines of evidence need to be considered: (1) enamel organ epithelia and dental papilla mesenchyme tissues contain stem cells during postnatal stages of life; (2) late cap stage and bell stage tooth organs contain stem cells; (3) odontogenic adult stem cells respond to mechanical as well as chemical "signals"; (4) presumably adult bone marrow as well as dental pulp tissues contain "odontogenic" stem cells; and (5) epithelial-mesenchymal interactions are pre-requisite for tooth regeneration. The authors express "guarded enthusiasm," yet there should be little doubt that adult stem cell-mediated tooth regeneration will be realized in the not too distant future. The prospects for tooth regeneration could be realized in the next few decades and could be rapidly utilized to improve the quality of human life in many nations around the world.     

Morphological study and stress analysis of Korean Mandibular second premolar

Tooth morphology has an important role in esthetical characteristics, functional performance and physiological action. Mandibular second premolars among teeth were taken for a close investigation with a micro-CT for a nondestructive measurement. Thus, the shape of the tooth was classified into a few partitions and measured by setting the reference axis to express internal and external morphology numerically. About a thousand tomographic images were obtained for each specimen and were reconstructed to a three-dimensional form. The lengths and widths of each dental tissue were measured from the three-dimensional form. In this tooth type, the width of dentine is comparatively larger than that of enamel, and buccal tissue of the crown is thicker than lingual tissue. Hence, buccal tissue could afford to withstand against exterior impact or stimulus. Finite element models were drawn up, applied with an ordinary occlusal force and analyzed to get the stress distribution. Thus it was shown that the irregular feature of the tooth is not useful only to masticating and pronouncing as well known, but that it is also suitable for protecting inner tissue by dispersing stress and delivering proper pressure to periodontal tissue to continue a physiological action. These results could be used for development of dental implants and clinical trials.     

Cutting teeth in the SEM

An SEM was used to observe and record dental tissues as they were being cut. Sound human deciduous and permanent teeth were stored in 70% ethanol until required, then soaked in water, superficially dried and screwed to the SEM specimen stage through small drill holes made when they were wet. Many specimens were frozen and studied at cryogenic temperatures so that they would not become dehydrated. Edges used to cut the teeth included steel and tungsten carbide fashioned to resemble clinical cutting instruments, and diamond ultramicrotome knives and burs. The cutting tools were held either in a micromanipulator or a rigid tool post clamped to the specimen stage. The finest control was obtained by moving the specimen with the usual stage controls. SEM was conducted at 3 or 5 kV using TV speed scanning on the uncoated samples. All experiments were video-taped. 3-D control was difficult with a mono image and a real-time stereo system was therefore developed. Continuous, flowing sections of enamel could be obtained using diamond knives to cut the prism-free, surface zone tangentially. Thin sections of dentine, cement and bone curled up as they were cut, thus demonstrating permanent deformation. Subsurface enamel always fractured as it was cut, either locally as the tissue passed over the knife edge or tore out beneath the plane of the knife or by larger fragments cleaving off at larger distances ahead of the knife. Appearances were characteristic of prism orientation with respect to cutting direction. No anisotropy of cutting behaviour was found with dentine or bone: These tissues only fractured when thicker sections were taken. The SEM methods employed here can be usefully applied in the study of other materials.     

On evaluation of wear resistance of tooth enamel and dental materials

A survey of the literature shows that in many studies on the wear resistance of tooth enamel or dental materials a large scatter of experimental data has been obtained when wear tests were performed at a fixed load. Despite the steady loading, wear conditions vary during sliding, since tooth enamel as well as dental materials have inhomogeneous structure. This leads to changes in contact interactions between sliding surfaces, and as a result, we get changes in the friction and wear behaviour of tested materials. This is why at the same loading the wear can be different. In this study, more reliable approach to evaluation of the wear resistance of human enamel and dental materials is proposed. The procedure is based on the correlation between the volumetric wear and the friction energy dissipated during sliding. The model can be useful to compare the wear resistance of different dental materials tested in different ambient conditions.     

Technical advances in the sectioning of dental tissue and of on‐section cross‐linked collagen detection in mineralized teeth

Immunohistochemical detection of cross‐linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on‐section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New®. It was possible to cut thin (1 μm) sections of mineralized teeth, and immunofluorescence characterization of cross‐linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was “patchy” and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on‐section protein detection but importantly for detecting cross‐linked fibrous collagens in both soft and mineralized tissue sections. Microsc. Res. Tech. 73:741–745, 2010. © 2009 Wiley‐Liss, Inc.     

Membranes,minerals, and proteins of developing vertebrate enamel

Developing tooth enamel is formed as organized mineral in a specialized protein matrix. In order to analyze patterns of enamel mineralization and enamel protein expression in species representative of the main extant vertebrate lineages, we investigated developing teeth in a chondrichthyan, the horn shark, a teleost, the guppy, a urodele amphibian, the Mexican axolotl, an anuran amphibian, the leopard frog, two lepidosauria, a gecko and an iguana, and two mammals, a marsupial, the South American short-tailed gray opossum, and the house mouse. Electron microscopic analysis documented the presence of a distinct basal lamina in all species investigated. Subsequent stages of enamel biomineralization featured highly organized long and parallel enamel crystals in mammals, lepidosaurians, the frog, and the shark, while amorphous mineral deposits and/or randomly oriented crystals were observed in the guppy and the axolotl. In situ hybridization using a full-length mouse probe for amelogenin mRNA resulted in amelogenin specific signals in mouse, opossum, gecko, frog, axolotl, and shark. Using immunohistochemistry, amelogenin and tuftelin enamel proteins were detected in the enamel organ of many species investigated, but tuftelin epitopes were also found in other tissues. The anti-M179 antibody, however, did not react with the guppy and axolotl enameloid matrix. We conclude that basic features of vertebrate enamel/enameloid formation such as the presence of enamel proteins or the mineral deposition along the dentin-enamel junction were highly conserved in vertebrates. There were also differences in terms of enamel protein distribution and mineral organization between the vertebrates lineages. Our findings indicated a correlation between the presence of amelogenins and the presence of long and parallel hydroxyapatite crystals in tetrapods and shark.     

Spectral Fluorescence Imaging for Quantitative Diagnosis and Mapping of Dental Fluorosis Affected Tooth Surfaces

Abstract

A new analytical method for quantitative diagnosis of dental fluorosis is suggested. The method is based on the spectral modifications in the fluorescence light emitted from affected enamel surfaces, relative to normal dental enamel. Imaging of the tooth surface is obtained using an imaging Fourier transform fluorescence spectrophotometer. This technique provides simultaneous fluorescence spectra at all pixels of the examined area. Images and the corresponding spectra were acquired at various tooth regions, and it was shown that normal, white opaque, brown discolored, and pitted tooth surfaces have different distinct spectral features which characterize the different degrees of the observed pathology of dental fluorosis. Criteria for quantitative assessment of the dental fluorosis stages were suggested. These were based on the measured spectral emission ratio at two wavelengths and comparison with the spectrum of normal enamel. The suggested parameters were validated against clinical observation of numerous samples affected by dental fluorosis at various pathological stages. Besides the introduction of the quantitative assessment, the method is suggested for detection of early changes in the characteristics of the tooth surface in dental fluorosis. The associated mapping capability allows for morphological characterization, which provides additional important clinical information.     

Microstructure and mineral composition of dental enamel of permanent and deciduous teeth

Purpose: This study evaluated and compared in vitro the microstructure and mineral composition of permanent and deciduous teeth's dental enamel. Methods: Sound third molars (n = 12) and second primary molars (n = 12) were selected and randomly assigned to the following groups, according to the analysis method performed (n = 4): Scanning electron microscopy (SEM), X‐Ray diffraction (XRD) and Energy dispersive X‐ray spectrometer (EDS). Qualitative and quantitative comparisons of the dental enamel were done. The microscopic findings were analyzed statistically by a nonparametric test (Kruskal‐Wallis). The measurements of the prisms number and thickness were done in SEM photomicrographs. The relative amounts of calcium (Ca) and phosphorus (P) were determined by EDS investigation. Chemical phases present in both types of teeth were observed by the XRD analysis. Results: The mean thickness measurements observed in the deciduous teeth enamel was 1.14 mm and in the permanent teeth enamel was 2.58 mm. The mean rod head diameter in deciduous teeth was statistically similar to that of permanent teeth enamel, and a slightly decrease from the outer enamel surface to the region next to the enamel‐dentine junction was assessed. The numerical density of enamel rods was higher in the deciduous teeth, mainly near EDJ, that showed statistically significant difference. The percentage of Ca and P was higher in the permanent teeth enamel. Conclusions: The primary enamel structure showed a lower level of Ca and P, thinner thickness and higher numerical density of rods. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Quantitative electron spectroscopic diffraction analyses of the crystal formation in dentine

Newly formed apatitic crystallites of different hard tissues consist, according to our investigations, of chains composed of nanometre-sized particles (islands, dots) arising at nucleating sites of the collagenous and noncollagenous matrix macromolecules. In dentine these islands coalesce rapidly in longitudinal direction to form needle-like crystallites which further coalesce to ribbon-like crystallites. We have concluded that the centre-to-centre distances between these islands represent the distances between the nucleating sites of the matrix macromolecules. We have applied energy-filtering transmission electron microscopy in the selected area electron diffraction mode at different stages of crystal formation in dentine and have obtained quantitative information of the degree of crystal disorder on the basis of the paracrystal theory. The fluctuation of the lattice plane distances in c -axis direction decreases, proceeding from the region near the dentine/predentine border to the dentine/enamel border.     

Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cells, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.     

Preservation of the negative image of tooth enamel with dental impression material enhances morphometric measurements of gingival overgrowth

Gingival overgrowth is a common health problem caused by genetic and environmental risk factors. Animal models for quantitative histological studies are needed to uncover genetic predisposition and dose-response data that might put individuals at increased risk for gingival disease. Gingival height, thickness, inflammation, and the degree of encroachment of gingiva over the tooth, are clinical measures of overgrowth; most of these parameters can be measured histologically, but in order to quantify gingival coverage of the tooth, the image of the crown must be present. Tooth and bone typically require decalcification for histology; thus, the tooth crown, a critical landmark, is lost. We describe a method for imaging the crown histologically, using impression materials applied to dissected mouse mandibles. Four dental alginates, three polyvinyl siloxanes, and one polyether and gelatin were used. The impression-material/mandibular tissue blocks were processed routinely. Polyvinyl siloxanes were incompatible with embedding resin; alginates, polyether and gelatin could be fixed, decalcified, embedded, and sectioned. Alginates and gelatin could be stained. Success in imaging the tooth crown varied with the preparation, but the alginates, polyether, and gelatin permitted a useful degree of measurement of exposed crown and enamel thickness, along with other morphometric parameters such as thickness of the dentin, lateral mandibular ramus, rete pegs, height of the gingiva, and volume density of vessels and inflammatory cells in the lamina propria. In conclusion, this new application for impression materials allows gingival coverage of tooth crown, as well as numerous other parameters to be measured for comparison with clinical data.     

Enamel alteration following tooth bleaching and remineralization

The purpose of this study was to compare the effects of professional tooth whitening agents containing highly concentrated hydrogen peroxide (with and without laser activation), on the enamel surface; and the potential of four different toothpastes to remineralize any alterations. The study was performed on 50 human molars, divided in two groups: treated with Opalescence^® Boost and Mirawhite^® Laser Bleaching. Furthermore, each group was divided into five subgroups, a control one and 4 subgroups remineralized with: Mirasensitive^® hap+, Mirawhite^® Gelle?, GC Tooth Mousse? and Mirafluor^® C. The samples were analysed by SEM/3D‐SEM‐micrographs, SEM/EDX‐qualitative analysis and SEM/EDX‐semiquantitative analysis. The microphotographs show that both types of bleaching cause alterations: emphasized perikymata, erosions, loss of interprizmatic substance; the laser treatment is more aggressive and loss of integrity of the enamel is determined by shearing off the enamel rods. In all samples undergoing remineralization deposits were observed, those of toothpastes based on calcium phosphate technologies seem to merge with each other and cover almost the entire surface of the enamel. Loss of integrity and minerals were detected only in the line‐scans of the sample remineralized with GC Tooth Mousse?. The semiquantitative EDX analysis of individual elements in the surface layer of the enamel indicates that during tooth‐bleaching with HP statistically significant loss of Na and Mg occurs, whereas the bleaching in combination with a laser leads to statistically significant loss of Ca and P. The results undoubtedly confirm that teeth whitening procedures lead to enamel alterations. In this context, it must be noted that laser bleaching is more aggressive for dental substances. However, these changes are reversible and can be repaired by application of remineralization toothpastes.     

Microtribological behaviour of human tooth enamel and artificial hydroxyapatite

The microtribological behaviour of human tooth enamel and artificial hydroxyapatite has been studied and compared in this paper using a nano-scratch tester, aiming to extend the understanding of the wear mechanism of enamel. Results showed that the microtribological behaviour of enamel differed obviously from that of artificial hydroxyapatite. Compared with the artificial hydroxyapatite, more significant plastic deformation happened to the enamel under low loads, but no brittle delamination mechanism occurred even under high loads. Enamel microstructure plays an extremely significant role in its microtribological behaviour. The results would be helpful for the development of new dental restorative materials.     

Erosion behaviors of human tooth enamel at different depth

In vitro erosion behaviors of human tooth enamel at different depth have been investigated in citric acid solution with pH 3.20. Erosion time of 3 min was used. The microtribological behaviors of eroded surfaces were also examined using nanoscratch technique. Results showed that erosion properties of enamel were closely associated with its location. Erosive substance loss increased from the outer to the interior enamel. Additionally, compared with the outer enamel, the influence of erosion on subsequent friction and wear behaviors appeared more significant in the interior enamel. Understanding of dental erosion would be useful for improving clinical management of toothwear.     

Osteoclast diseases

Osteoclasts are the only cells capable of resorbing mineralised bone, dentine and cartilage. Osteoclasts act in close concert with bone forming osteoblasts to model the skeleton during embryogenesis and to remodel it during later life. A number of inherited human conditions are known that are primarily caused by a defect in osteoclasts. Most of these are rare monogenic disorders, but others, such as the more common Paget's disease, are complex diseases, where genetic and environmental factors combine to result in the abnormal osteoclast phenotype. Where the genetic defect gives rise to ineffective osteoclasts, such as in osteopetrosis and pycnodysostosis, the result is the presence of too much bone. However, the phenotype in many osteoclast diseases is a combination of osteosclerosis with osteolytic lesions. In such conditions, the primary defect is hyperactivity of osteoclasts, compensated by a secondary increase in osteoblast activity. Rapid progress has been made in recent years in the identification of the causative genes and in the understanding of the biological role of the proteins encoded. This review discusses the known osteoclast diseases with particular emphasis on the genetic causes and the resulting osteoclast phenotype. These human diseases highlight the critical importance of specific proteins or signalling pathways in osteoclasts.