Elsevier

Dental Materials

Volume 26, Issue 1, January 2010, Pages e1-e10
Dental Materials

3D variations in human crown dentin tubule orientation: A phase-contrast microtomography study

https://doi.org/10.1016/j.dental.2009.09.007Get rights and content

Abstract

Objectives

Tubules dominate the microstructure of dentin, and in crowns of human teeth they are surrounded by thick mineralized peritubular cuffs of high stiffness. Here we examine the three-dimensional (3D) arrangement of tubules in relation to enamel on the buccal and lingual aspects of intact premolars and molars. Specifically we investigate the angular orientation of tubules relative to the plane of the junction of dentin with enamel (DEJ) by means of wet, non-destructive and high-resolution phase-contrast (coherent) tomography.

Methods

Enamel capped dentin samples (n = 16), cut from the buccal and lingual surfaces of upper and lower premolar and molar teeth, were imaged in water by high-resolution synchrotron-based phase-contrast X-ray radiography. Reconstructed 3D virtual images were co-aligned with respect to the DEJ plane. The average tubule orientation was determined at increasing distances from the DEJ, based on integrated projections onto orthogonal virtual planes. The angle and curl of the tubules were determined every 100 μm to a depth of 1.4 mm beneath the DEJ.

Results

Most tubules do not extend at right angles from the DEJ. Even when they do, tubules always change their orientations substantially within the first half-millimeter zone beneath the DEJ, both on the buccal and lingual aspects of premolar and molar teeth. Tubules also tend to curl and twist within this zone. Student t-tests indicate that lower teeth seem to have greater tilts in the tubule orientations relative to the DEJ normal with an average angle of 42° (±2.0°), whereas upper teeth exhibit a smaller change of orientation, with an average of 32° (±2.1°).

Significance

Tubules are a central characteristic of dentin, with important implications on how it is arranged and what the properties are. Knowing about the path that tubules follow is important for various reasons, ranging form improving control over restorative procedures to understanding or simulating the mechanical properties of teeth. At increasing depths of dentin beneath enamel, tubules are significantly tilted relative to the DEJ norm, which may be important to understand clinical challenges such as sensitivity, effectiveness of bonding techniques or prediction of possible paths for bacterial invasion. Our data show dissimilar average tubule angles of upper versus lower teeth with respect to the DEJ which presumably contributes to different shear responses of the tissue under function. The degree to which this may warrant improved restoratives or new adhesive techniques to enhance adhesive restorations merits further investigation.

Introduction

Dentin in human teeth has been extensively researched for more than 150 years [1] and in recent decades, details of the microstructure have been augmented by extensive studies of its material characteristics. As a result, both elastic and plastic/failure attributes of this biological material are rather well understood in relation to the tissue organization (see overview in [2]). And yet, open questions remain as to what in the microstructure contributes most to the tissue properties, and in particular how the complex 3D design is able to withstand the trying conditions of the mouth for extensive periods of time. This is remarkable, because dentin is a variant of the mineralized collagen based tissues [3] that does not remodel or self-fix, such that biological turnover is relatively insignificant. And in light of the fact that man-made restorations do not perform nearly as well as dentin in vivo (despite having excellent properties), it is prudent to assume that there are details of the 3D organization of features in dentin that we still do not fully understand. Dentin attributes are routinely considered with respect to tubules, and in this study, we report on the orientations of tubules relative to the DEJ plane on the buccal and lingual aspects of human premolar and molar teeth.

As a member of the bone family of materials [3] and at the lowest organizational level dentin is composed of mineral, organic macromolecules (mostly collagen) and water. The mineral, a calcium phosphate salt (dahllite), accounts for about 65% of the weight. Similar to bone, the mineral appears in the form of small crystals of carbonated hydroxyl-apatite, sized ∼36 nm × 25 nm × 4 nm [4], [5]. The mineral embeds the organic macromolecules that account for about 20–25% of the weight and appear mostly as a felt of thin (∼100–200 nm thick) highly cross-linked type I collagen fibers (about 90%) [6]. Other organic components include non-collagenous phosphorylated/non-phosphorylated proteins, small amounts of proteoglycans, neutral and acidic mucopolysaccharides and lipids.

At a higher organizational level, dentin is a porous material, densely perforated by more-or-less parallel tubules that lead inwards towards the pulp chamber. Despite being a thick sensitive biological tissue, dentin is a-cellular. The tubules however, often contain protrusions or extensions of living cells—the odontoblasts [7], [8]. These cells are responsible for excreting tissue products that assemble into dentin [1] while mineralizing along a front surrounding the cellular protrusions and continuously moving inwards towards the pulp and down the roots. After tooth formation is complete, the very same odontoblasts continue to produce dentin at a slow but steady pace within the pulp chamber and root canals, forming a lower quality and less organized tissue. Dentin in vital teeth is thus in fact part of a dentin–pulp complex [9] that is both sensitive and reactive to the tooth environment.

The tissue created by the odontoblasts is in effect a complex three-dimensional arrangement of several forms of dentin, most notably pertitubular dentin (PTD) and intertubular dentin: While the former (PTD) is known to be highly mineralized and contains no collagen, the latter is made of mineralized collagen fibers that presumably lie in incremental layers approximately parallel to the pulp wall, at right angles to the tubules. PTD lines many of the tubules (Fig. 1), mainly in the crown and is formed at the time when the bulk of dentin is being laid down – during tooth formation [10]. PTD differs from the mineral precipitates that may collect in tubules – particularly in older dentin, where tubules become occluded or sclerotic over time [1]. It creates dense collars with internal diameters of about one micrometer for most of the tubule length. The PTD surrounded tubules extend up to several millimeters along their long axis as they follow an S shape path inwards towards the pulp. It is deep in the bulk of the crown that PTD is thickest (>1 μm). Near the dentino–enamel junction (DEJ) in the crown (or the cemento–dentinal junction in the root) in the outer regions of dentin, close to where tubules branch and originate (with diameters of less than 0.5 μm [11], [12]) PTD is thin or missing. On the other end in deep dentin near the pulp, tubules become tightly packed as they converge together, with their internal diameters exceeding 2 μm [7], [9].

The uniform and highly aligned distribution of tubules suggests that dentin should be a very anisotropic tissue, deforming differently when loaded along versus across tubule orientations. But curiously, little or negligible elastic anisotropy has been found [13]. Dozens of experiments performed over many years and for a varying range of sample sizes and measurement techniques have shown that dentin exhibits only hints of anisotropy. And yet nanoindentation has revealed large differences in the stiffness of PTD as compared with intertubular dentin—the former amounting to 30 GPa while the latter measuring only 15–20 GPa [14]. With such a ratio one might expect to find a lower modulus when loading dentin samples orthogonal to the tubule direction (effectively densifying the tubules that are embedded in less-stiff intertubular dentin), as compared with loading along the tubule orientations. This however is not seen, and Kinney et al. [15] developed a micromechanics model showing that despite increasing thickness and density of the PTD, the tubular structures on average do not reinforce dentin and hence they do not impose mechanical anisotropy: dentin properties are thus governed by the intertubular dentin. Few studies however, such as the one by Palamara et al. [16], reported small differences in moduli along versus across the main tubule orientations when measured in compression (E = 10.7 and 11.9 GPa, respectively). Using resonant ultrasound spectroscopy, Kinney et al. [13] concluded that an orthogonal arrangement of collagen fibrils with respect to tubules renders human dentin approximately 10% anisotropic [17]. They suggested that dentin has a Young's modulus of 23.2 GPa along the tubules versus 25.0 GPa across the tubules. Arola and Reprogel [18] used bending experiments to provide additional evidence to the existence of some elastic anisotropy related to tubule orientation: they reported flexural moduli of 15.5 GPa when bending bar samples where tubules run along the long bar axis, versus 18.7 GPa when bending samples with tubules orthogonal to the long bar direction. Reduced stiffness was found in the near-DEJ zone [11], [27], [28] where collagen fibers run orthogonal to enamel and parallel to the tubules. In all these studies however, tubule orientations were determined or inferred from observations on the edges of the samples, but no direct link between tubule orientations and elastic properties was shown.

Unlike elastic properties, anisotropy with respect to tubule orientation has been rather clearly demonstrated for failure properties of human dentin. Rasmussen et al. [19] demonstrated that the work to fracture across tubules was about half that of the work to fracture along the tubule orientation. A dependence of shear strength on dentin site and tubule orientation within the crown was clearly demonstrated by Watanabe et al. [20] and tensile strength anisotropy was reported by Carvalho et al. [21] and other workers. Arola and Reprogel [18] demonstrated significantly reduced strains at fracture (about 0.005 m/m) along tubule orientations compared with higher strain values across tubule orientation (0.015 m/m) and reported increased flexural strength across the tubules (∼160 MPa)- 30% higher than the strength along the tubules. They also noted a decay of strength with increasing tubule density nearing the pulp. These and other studies of fracture toughness and fatigue have demonstrated that failure in dentin occurs much more easily across tubules than along their trajectory (see for example [22], [23], [24], [25], [26]), possibly due to increased brittleness when tubules are pulled or compressed along the long axis.

The plastic properties of dentin suggest that different tubule orientations in human dentin may lead to significant variations in the mechanical properties. These orientations may also have an indirect effect on the stiffness. Tubules beneath enamel are thought to extend orthogonal to the DEJ ‘plane’ and they then follow a somewhat winding S shaped path deep into the bulk [1]. We propose that a better understanding of dentin as a structure and specifically its mechanical properties may arise from knowing more about the 3D course that the tubules occupy when passing through the tissue. In particular, large – mm sized test samples – may have unknown tubule orientations. Recent methods of non-destructive high-resolution and partially coherent X-ray imaging allow exploring tubule orientation variations deep in the dentin bulk. One excellent method of achieving this is by microtomography.

High-resolution X-ray microradiography and microtomography have become important investigation tools, useful for imaging minute details spanning less than 10 μm within small representative (millimeter sized) samples. Absorption is the primary interaction of interest, because attenuation of the X-ray signal is linked to important sample attributes: geometry, density and composition. Briefly, for each of many projection images taken from equally spaced angles around the sample (at least 180° for parallel beam geometry, usually obtained by rotating the sample) it is possible to obtain the ratio between sample attenuated intensities and non-attenuated intensities in images where the sample is absent. For tomography these ratios are used for numerical backprojection, so as to create 3D images of the volume which represent the sample structure. Additional details about tomography and methods of reconstruction may be found in textbooks (for example see [29]) as well as in many online references.

Inherent to high-resolution X-ray absorption imaging is the problem of low signal to noise ratios. Variations in the source intensity and stability, detector response limitations and faults, system vibrations and thermal fluctuations all contribute to lack of precision and a reduction in the clarity of features in each projected radiograph. This results in deteriorated 3D reconstructions and the emergence of artifacts. Furthermore, absorption imaging has limited use when we consider that most biomaterials are made of light elements that induce little or negligible attenuation contrast between adjacent materials or structures. The light elements are almost transparent to X-rays and they contribute significantly more to scattering than to absorption, which reduces the signal to noise ratio dramatically. The outcome of all this is that the small structural details that exist in dentin bring about similar and indistinguishable attenuation effects in most absorption radiographs, and consequently standard or ‘regular’ microtomography is of little use for the study of the thin dentinal tubules and their 3D orientations in dentin.

High energy hard X-ray sources such as those found in 3rd generation synchrotron (electron acceleration) facilities offer extremely high brilliance at a range of energies that is relevant for the study of mineralized tissues (20–40 keV) [30]. Large propagation distances (tens of meters) between the X-ray source and detectors in these huge machines entail some laser-like attributes to the X-rays: they become partially coherent, such that constant phase relations are found over meaningful distances compared to the sample dimensions [31]. This leads to the appearance of interference patterns/fringes in radiographs so that either constructive (brighter) or destructive (darker) spots/lines are seen. Such fringes arise due to the difference in interaction of the radiation with matter found on opposing sides of edges, voids and discontinuities. This difference results in an overlapping of mutually coherent X-ray fields on the detector, positioned far behind the sample, and this difference is a consequence of the beam passing through different path densities within the sample microstructure. Note that even between sample features composed of light elements and even if these features are of micron or sub-micron dimensions, significant path density differences arise, seen as edge enhancements and inhomogeneity accentuation in the radiographs.

In this paper we report on measurements and findings of tubule orientation variations that are seen in a zone of 1.4 mm beneath the DEJ on the buccal and lingual sides of molar and premolar teeth, using phase-contrast tomography. Phase-contrast imaging of this type is not a new concept: the ability to project and produce slightly enlarged phase-enhanced silhouettes of edges in objects was originally used for inline holography and electron microscopy, pioneered by Gabor [32] and others in the late 1940s. However, with current X-ray instrumentation and when coupled with tomographic reconstruction, the technique brings about contrast enhancement even between very small or chemically similar material phases. As a result, it is possible to visualize the 3D structure and distribution of features sized much less than a micrometer [33] so as to reveal sub-micron details [34]. In particular dentin tubules, even when immersed in water, may be clearly visualized, as long as destructive and constructive interference patterns do not cancel each other out [35].

Section snippets

Tooth sample preparation

Samples were cut from the mid-buccal and mid-lingual surfaces of 8 intact permanent teeth, extracted from young people in private clinics in the greater Berlin area (Germany) in accordance with conditions set by the ethical review board of the Berlin Charité dental school. Four molars (2 upper and 2 lower wisdom teeth) extracted from young adults and 4 premolars (2 upper and 2 lower) extracted from teenagers during orthodontic treatment were used. Samples approximately 2 mm × 2 mm × 3 mm were manually

Results

Fig. 5 shows the average tubule tilt, away from the Z-axis (normal to the DEJ) as a function of distance from the DEJ. Note the wide distribution of angles of relative tilt within the first 500 μm beneath enamel. Note also, that beyond a depth of 800 μm all tubules in all samples are significantly tilted relative to the DEJ plane normal. The figure highlights samples belonging to the lower versus upper jaws; for depths of 600–1000 μm, a significant difference in the average tubule orientation φ of

Discussion and conclusions

Dentin is dominated by tubules that render the structure porous. Recent high-resolution 3D imaging methods now allow to take closer looks at how these micrometer features relate to the tooth as a whole, possibly shedding light on how it functions or why it fails. Much evidence has accumulated over the years suggesting that the mechanical properties of dentin vary in a manner that is linked with tubule orientations. This link is however rather indirect, related to the fact that tubules in dentin

Acknowledgments

The authors gratefully acknowledge financial support from the Max-Planck Society. X-ray measurements were performed on BAMline microCT imaging beamline in BESSY, the electron storage-ring of the Helmholtz-Center for Materials and Energy in Berlin, and we are grateful for the support of Dr. Heinrich Riesemeier during these experiments.

References (48)

  • J.D. Wood et al.

    Mapping of tooth deformation caused by moisture change using Moiré interferometry

    Dent Mater

    (2003)
  • W. Gorner et al.

    BAMline: the first hard X-ray beamline at BESSY II

    Nucl Instrum Methods Phys Res A: Accel Spectrom Detect Assoc Equip

    (2001)
  • A. Rack et al.

    High resolution synchrotron-based radiography and tomography using hard X-rays at the BAMline (BESSY II)

    Nucl Instrum Methods Phys Res A: Accel Spectrom Detect Assoc Equip

    (2008)
  • J. Vlassenbroeck et al.

    Software tools for quantification of X-ray microtomography

    Nucl Instrum Methods Phys Res A: Accel Spectrom Detect Assoc Equip

    (2007)
  • M.C. Cagidiaco et al.

    Mapping of tubule and intertubule surface areas available for bonding in class V and class II preparations

    J Dent

    (1997)
  • A. Nanci

    Ten cate's oral histology: development, structure and function

    (2003)
  • P. Zaslansky

    Dentin

  • S. Weiner et al.

    The material bone: structure-mechanical function relations

    Ann Rev Mater Sci

    (1998)
  • L. Schroeder et al.

    High resolution transmission electron microscopy of adult human peritubular dentine

    Cell Tissue Res

    (1985)
  • M. Goldberg et al.

    Dental mineralization

    Int J Dev Biol

    (1995)
  • G. Goracci et al.

    Terminal end of human odontoblast process: a study using SEM and confocal microscopy

    Clin Oral Invest

    (1999)
  • D.H. Pashley

    Dynamics of the pulpo–dentin complex

    Crit Rev Oral Biol Med

    (1996)
  • S. Takuma et al.

    Structure and development of peritubular matrix in dentin

    J Dent Res

    (1966)
  • S.J. Jones et al.

    Ultrastructure of dentin and dentinogenesis

  • Cited by (47)

    • 4D microstructural changes in dentinal tubules during acid demineralisation

      2021, Dental Materials
      Citation Excerpt :

      Imaging 3D dentine structures using SXM, non-destructively made it possible to resolve the ITD and PTD phases. Although SXM has been used to image dentine tubules with submicron resolution, distinctly resolving PTD and ITD has not always been possible due to insufficient phase contrast [23–25] or multiple scans required for high quality tubule reconstruction [26] which prevents capturing fast chemical reactions. In this study, the nominal resolution available was able to resolve most of the tubules in a single scan with some contrast between PTD and ITD.

    • Multiscale micromechanical modeling of the elastic properties of dentin

      2019, Journal of the Mechanical Behavior of Biomedical Materials
    • Fabrication and characterisation of a novel biomimetic anisotropic ceramic/polymer-infiltrated composite material

      2018, Dental Materials
      Citation Excerpt :

      Throughout a lifetime, enamel can endure the forces of mastication over millions of cycles [12,13]. By contrast, dentine is a porous mineralised structure that is composed of 70% hydroxyapatite, 20% collagen and 10% water by weight [14]. Because dentine is very tough, and enamel is much harder and more brittle, they need to be joined together to provide a biomechanically compatible system.

    • Numerical and analytical investigation of irrigant penetration into dentinal microtubules

      2017, Computers in Biology and Medicine
      Citation Excerpt :

      By solving the unsteady form of concentration equation numerically, Verhaagen et al. [19] showed that at the end of a tubule, concentration reaches 86% of the initial concentration after 10 min. In reality, dentinal tubules have a conical shape [20] and have S-shape curvatures along their path in the coronal parts that changes gradually to straight shape in the apical parts [21]. In this study, the results of modeling of the time dependent irrigant penetration into more realistically shaped tubules that are located in different parts of the root canal were presented.

    View all citing articles on Scopus
    View full text