4D microstructural changes in dentinal tubules during acid demineralisation
Graphical abstract
Introduction
Dental erosion is the irreversible loss of dental hard tissues due to chemical dissolution by acids of non-bacterial origin [1]. The source of acid can be intrinsic (gastric acid, i.e. hydrochloric acid) [2], or extrinsic (dietary acids, medication or the environment) [3]. For the general populace, the primary source of acids are from acidic foods and beverages containing various acids including acetic, citric, lactic, malic and tartaric [4]. The inorganic composition of mineralised dental tissues mostly consists of a substituted form of hydroxyapatite (HAp), described as a calcium-deficient carbonated HAp with some fluoride [5]. In the oral environment, HAp in the dental tissues is in ionic homeostasis between demineralisation and remineralisation, represented by the simplified chemical equation:where demineralisation is the reaction going from left to right [6]. This homeostasis is maintained by hydroxycarbonate and phosphate buffers and inorganic Ca2+ and F− ions from saliva. When exposed to acids, the low pH disrupts the ionic balance and results in the loss of minerals in the teeth. In response, salivary flow is increased to deliver more buffers and ions that neutralise the acid and allow for remineralisation to occur. However, this remineralisation is limited and ultimately results in net mineral loss depending on the severity of the acid exposure [7].
Dentine forms the main bulk of the tooth, supporting enamel that encapsulates the crown and cementum as the superficial layer on the root of the tooth. At the microscopic scale, dentine is structurally anisotropic with numerous tubules containing odontoblast processes [8]. In between these tubules there is intertubular dentine (ITD), composed of plate-like HAp crystallites (3 nm thick, 60 nm long) embedded in a dense matrix of collagen fibrils. Around the lumen of the tubules, peritubular dentine (PTD) appears more mineralised than ITD and consists of isodiametric HAp crystallites, (∼25 nm in diameter) embedded in a non-collagenous matrix of phospholipids and proteins [9]. Although it is generally inaccessible to the oral environment, dentine can become exposed to acids in the mouth after enamel or cementum have been lost due to dental attrition, abrasion, erosion, or following dental fracture. Despite having a higher degree of mineralisation, the finer-sized HAp crystallites provides a higher surface-to-volume ratio that makes PTD more susceptible than ITD to acid dissolution [10]. As such, the demineralisation process starts in PTD, causing dentinal tubules to widen and eventually affects ITD, leaving a superficial layer of demineralised collagen [11] which can be broken down by bacteria and host enzymes, such as matrix metalloproteinases [12].
Past observational studies of dentinal tubules at the micro- and nanoscales with conventional and advanced microscopy techniques such as scanning electron (SEM), [13], transmission electron (TEM) [14] and atomic force microscopy (AFM) [15] have allowed observation of the microstructure and mechanical properties of PTD and ITD after ex situ acid demineralisation. Those techniques revealed important characteristic changes in PTD and ITD, although ex situ experiments captured information from the end-result, not during the process of demineralisation and lacked the detail of time-resolved analysis. In addition, TEM requires destructive preparations of the samples which means that is it impossible to make time-resolved observations of the same tubules during demineralisation and SEM does not allow real time in situ experimentation. More insightful observations could be made from 3D studies of the dentinal structure collected in a time-resolved manner (simply referred to as 4D studies). which have been performed using AFM [[16], [17], [18], [19], [20]]. However, observations were limited to a depth of ∼2 μm from the sample surface as PTD quickly demineralises beyond the reach of the AFM probe which then is unable to examine the mineralised ITD beyond the demineralised collagen matrix at the sample surface. Hence, that technique is only capable of characterising the initial stages of dentine demineralisation. X-ray micro-computed tomography (XMT) is non-destructive and enables observation of large 3D volumes, allowing the progression of demineralisation to be monitored at depths that are hundreds of micrometres beyond the capabilities of AFM. XMT has been used to visualise dentinal tubules with submicron resolution and reasonable contrast between ITD and PTD [[23], [24], [25], [26]]. XMT was also used to study interrupted ex situ dentine demineralisation (where a sample was demineralised for a period outside the instrument before scanning and following this was taken out and demineralised further and the process repeated.) [14,27] to measure and map mineral densities although without sufficient spatial resolution to observe structural changes of dentinal tubules. Therefore, it has not been possible to make complete observations of the 3D spatio-temporal changes occurring in ITD and PTD during demineralisation which are essential for understanding the processes involved.
High-speed synchrotron X-ray microtomography (SXM) is capable of high spatial and temporal resolutions, compared with other X-ray techniques and is ideally suited for time-resolved studies. The authors therefore propose this technique as a method for 4D dentine demineralisation studies. The present study aims to identify demineralisation rates, structural and dimensional changes, by means of time-resolved, submicron resolution SXM to determine its suitability for such studies. To the authors’ best knowledge, this is the first investigation of continuous PTD and ITD demineralisation using high-speed SXM to reveal 3D microstructural dentine changes with time.
Section snippets
Sample preparation
Non-carious human third molars, extracted at the Birmingham Dental School and Hospital for therapeutic reasons (BCHCDent332.1531.TB, REC Ref.:14/EM/1128), were fixed in 10% neutral buffered formalin (Sigma Aldrich, UK). Samples (∼0.8 × 0.8 × 5 mm dentine cuboids) were prepared using a low-speed diamond bone saw (Isomet™, Buehler, UK) by first cutting a ∼1 mm thick section ±0.5 mm either side of the centre of the occlusal surface, cutting from the crown to the root. A further two cuts were made
3D visualisation
Fig. 2 shows the segmented tubules at time points from 15 to 90 min, with an additional rendering at 180 min. Above the demineralised surface, bubbles formed by a combination of the dissociation of H2O molecules (due to X-ray exposure) and CO2 generated from the reaction between acid and carbonated HAp and had a similar contrast to the tubules. Immediate observations revealed that the depth of heavily demineralised dentine varied across samples, with Sample#1 demineralised the most and Sample#2
Discussion
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], [24], [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
Conclusion
High-speed SXM has been shown to be suitable method for measuring the dissolution rates of ITD and PTD whilst providing the ability to study relatively large volumes of dentine. Although AFM is more convenient, the ability to monitor the progressive demineralisation in dentine to a greater depth is more worthwhile.
In summary, observations made with this technique, such as those made in the present study, could lead to the establishment of reliable numerical models of dentine demineralisation
Acknowledgements
The authors give thanks to Dr Jonathan James from the University of Birmingham, for his help in preparing samples used in this work. The authors would also like to thank Dr Shashidhara Marathe, and Dr Kazimir Wanelik, from the I13 beamlines at Diamond Light Source (DLS), for their support and assistance with data acquisition, reduction and analysis. DLS is also acknowledged for providing access to the I13-2 facilities under the allocation MT20155. The EPSRC project (EP/S022813/1) “Understanding
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