Bovine and equine peritubular and intertubular dentin

doi:10.1016/j.actbio.2014.05.027

Acta Biomaterialia

Volume 10, Issue 9, September 2014, Pages 3969-3977

https://doi.org/10.1016/j.actbio.2014.05.027 Get rights and content

Abstract

Dentin contains 1–2 μm diameter tubules extending from the pulp cavity to near the junction with enamel. Peritubular dentin (PTD) borders the tubule lumens and is surrounded by intertubular dentin (ITD). Differences in PTD and ITD composition and microstructure remain poorly understood. Here, a (∼200 nm)², 10.1 keV synchrotron X-ray beam maps X-ray fluorescence and X-ray diffraction simultaneously around tubules in 15–30 μm thick bovine and equine specimens. Increased Ca fluorescence surrounding tubule lumens confirms that PTD is present, and the relative intensities in PTD and ITD correspond to carbonated apatite (cAp) volume fraction of ∼0.8 in PTD vs. 0.65 assumed for ITD. In the PTD near the lumen edges, Zn intensity is strongly peaked, corresponding to a Zn content of ∼0.9 mg g⁻¹ for an assumed concentration of ∼0.4 mg g⁻¹ for ITD. In the equine specimen, the Zn K-edge position indicates that Zn²⁺ is present, similar to bovine dentin (Deymier-Black et al., 2013), and the above edge structure is consistent with spectra from macromolecules related to biomineralization. Transmission X-ray diffraction shows only cAp, and the 00.2 diffraction peak (Miller–Bravais indices) width is constant from ITD to the lumen edge. The cAp 00.2 average preferred orientation is axisymmetric (about the tubule axis) in both bovine and equine dentin, and the axisymmetric preferred orientation continues from ITD through the PTD to the tubule lumen. These data indicate that cAp structure does not vary from PTD to ITD.

Graphical abstract

Introduction

Dentin is a porous mineralized tissue providing toughness to the dental organ. Tubules run through dentin, extending from near the dentinoenamel junction (DEJ) to the pulp cavity. Odontoblasts produce these long open channels as they move away from the DEJ during tooth development, and the tubules (typically 1–2 μm in diameter spaced 5–10 μm apart) are filled with odontoblastic processes or their remnants, i.e., fluid or soft tissue. The dentin surrounding these tubules contains two phases: intertubular dentin (ITD), which forms between the odontoblastic processes (tubules), and peritubular dentin (PTD), which grows after ITD mineralizes and around the circumference of the tubule and into its lumen. ITD makes up most of the volume of dentin and is a composite consisting of a matrix of collagen fibrils discontinuously reinforced with nanoplatelets of carbonated hydroxyapatite (cAp), making it strong but tough [1]. PTD is more highly mineralized than ITD [2], [3], [4] but contains little to no collagen [4], [5], [6], making it significantly harder and stiffer than the ITD. It remains unclear exactly how cAp in ITD and in PTD are related to each other and how PTD forms adjacent to ITD.

Earlier, diffraction and fluorescence mapping around bovine dentin tubules was reported in 1 μm thick samples [7], [8]. A 250 or 200 nm diameter beam was scanned across the specimens and mapped the variation of Ca and Zn fluorescent intensity and of the cAp crystallographic preferred orientation around tubules. Crystallographic preferred orientation did not change between positions remote from and adjacent to the tubule. Further, these studies found that Zn²⁺ concentrated around the tubule lumens but enhanced Ca signal near the tubules was not clearly observed, i.e., hypermineralization characteristic of PTD vs. ITD [2], [3], [9].

The earlier studies [7], [8] had limitations. Diffracted intensities, originating from the entire sample thickness, were very weak because there were relatively few nanoplatelets irradiated at each position (∼5 × 10³), and noisy maps resulted. Further, data from 1 μm mineralized samples may be dominated by surface-related sample preparation artefacts. Increasing the sample thickness to 20–30 μm does not appreciably affect absorption of 10 keV X-ray photons but increases the number of platelets sampled to ∼1 × 10⁵, improves counting statistics by more than a factor of four (for the same counting time) over those reported earlier [7], [8] and provides signals dominated by material further than 1 μm from the surface.

A consequence of increasing the sample thickness is that lateral “averaging” of structural details can result. For 200 nm mapping, a 1 μm thick sample possesses an aspect ratio (thickness to beam width) of 5, and a 20 μm thick sample has an aspect ratio of 100. Mapping in ∼20 μm thick specimens would be problematic, therefore, if closely spaced, sharply defined structures were present instead of structures which change gradually with lateral position and which are relatively uniform through the specimen thickness. Earlier tubule mapping (∼200 nm wide X-ray beam, ∼1 μm thick specimen) showed, however, that fluorescent and diffracted signals varied smoothly around tubules [7], [8]. It should, therefore, be possible to map the surroundings of tubules running through the thicker specimens and perpendicular to the surfaces, and demonstrating this is the first goal of the studied reported below.

The earlier studies [7], [8] were also limited in the number and locations of tubules studied, in the number of teeth observed (one incisor and one molar), in the species examined (Bos taurus, breed black Angus) and in the age of the animal (12–18 months). The study reported below examines tubules in dentin from additional animals and an additional species (Equus ferus). The earlier studies also did not clearly identify enhanced levels of Ca where PTD was expected, and, given that the presence of PTD can be quite variable within bovine teeth, it is unclear whether these results reflect an intrinsic limitation of using 10 keV photons to excite Ca fluorescence (Kα energy of 4.03 keV) to look for potentially small changes in Ca on top of an already large signal from ITD or whether the tubules sampled did not contain prominent PTD. Therefore, the third goal of this study is to identify and characterize PTD by mapping cAp diffracted intensity and crystallographic preferred orientation and Ca and Zn fluorescent intensity around tubules in 15–30 μm thick bovine and equine samples.

Section snippets

Sample preparation

Erupted bovine pre-molars were extracted from two animals 12–18 months in age and cast in LR White (Electron Microscopy Sciences, Hatfield, PA). An equine incisor from a 10-year-old animal was prepared in a similar way. Cuts ∼125–150 μm thick were made perpendicular to the nominal tubule axes using an Isomet 1000 wafering saw (Buehler, Lake Bluff, IL). These sections were polished by hand to thicknesses of ∼15–35 μm using 500 grit SiC polishing paper placed between two glass microscope slides;

Results

Fig. 1 shows transmission optical micrographs of areas of the specimens within which the maps of Fig. 2 were obtained. The tubules are more closely spaced than in the earlier studies [7], [8], and the tubules have circular cross-sections, which means that they are close to perpendicular to the faces of the 15–30 μm thick plates of dentin. The tubule diameters within the fields of view of Fig. 1 appear slightly larger than 2 μm although, at this magnification and through this specimen thickness,

Discussion

In the optical micrographs of the samples mapped in Fig. 2, the number densities of tubules were 5–6 × 10⁴ mm⁻² (Fig. 1a, bovine) and ∼4 × 10⁴ mm⁻² (Fig. 1b, equine). In the bovine dentin mapped earlier, there were 4.3 × 10⁴ tubules mm⁻² [7]. For comparison, Schilke et al. [14] measured 2.1–4.7 × 10⁴ tubules mm⁻² within bovine dentin, and a micrograph of equine incisor dentin of Muylle et al. [15] showed 3.1 × 10⁴ tubules mm⁻². The spacings between tubules derived from the diffraction and fluorescence maps are

Conclusions and future directions

The present study uses specimens substantially thicker than those used before [7], [8], and this does not appear to affect accuracy but greatly increases diffracted intensity and improves sample handling. With thicker specimens, most of the diffracting volume is well away from the polished surfaces and is unaffected by sample preparation. The authors are quite fortunate to obtain 3–4 days of beam time for the mapping experiments each of three scheduling cycles annually, and this, coupled with

Disclosures

The authors have no conflict of interest to report.

Acknowledgements

The authors thank Dr M.C. Stewart (Veterinary College, University of Illinois-Urbana-Champaign) for providing the horse incisor. The research was supported by NICDR grant DE001374 (to A.V.). The funding source had no role in the planning, execution or reporting of this study. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

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^☆: Part of the Biomineralization Special Issue, organized by Professor Hermann Ehrlich.

¹: Current address: Department of Orthopaedic Surgery, Washington University in St Louis, 660 S. Euclid Ave, Campus Box 8233, St Louis, MO 63110, USA.

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Bovine and equine peritubular and intertubular dentin☆

Abstract

Graphical abstract

Introduction

Section snippets

Sample preparation

Results

Discussion

Conclusions and future directions

Disclosures

Acknowledgements

J Struct Biol

J Struct Biol

J Struct Biol

Acta Biomater

J Mech Behav Biomed Mater

Arch Oral Biol

J Ultrastruct Res

Arch Oral Biol

Micron

J Mol Biol

Acta Biomater

J Biol Chem

Arch Oral Biol

Oral histology: development, structure and function

Structure and development of the peritubular matrix in dentin

J Dent Res

The distribution of peritubular matrix in human coronal dentin

J Morphol

Peritubular dentin, a vertebrate apatitic mineralized tissue without collagen: role of a phospholipid–proteolipid complex

Calcif Tissue Int