Leaching and cytotoxicity of bismuth oxide in ProRoot MTA - A laboratory investigation.

AIM: The present study examined the leaching and cytotoxicity of bismuth from ProRoot MTA and aimed to identify whether bismuth leaching was affected by the cement base and the immersion regime used. METHODOLOGY: The leaching profile of bismuth was examined from ProRoot MTA and compared with hydroxyapatite containing 20% bismuth oxide as well as hydroxyapatite and tricalcium silicate to investigate whether bismuth release changed depending on the cement base. Bismuth leaching was determined after 30 and 180 days of ageing immersed in Dulbecco's modified Eagle's medium (DMEM) using mass spectroscopy (ICP-MS). The media were either unchanged or regularly replenished. The pH, surface microstructure and phase changes of aged materials were assessed. Wistar rat femoral bone marrow stromal cells (BMSCs) and cutaneous fibroblasts were isolated, cultured and seeded for cell counting (trypan blue live/dead) after exposure to non-aged, 30- and 180-days-aged samples in regularly replenished DMEM. Aged DMEM in contact with materials was also used to culture BMSCs to investigate the effect of material leachates on the cells. Gene expression analysis was also carried out after direct exposure of cells to non-aged materials. Differences between groups were statistically tested at a significance level of 5%. RESULTS: All materials exhibited alterations after immersion in DMEM and this increased with longer exposure times. The bismuth leached from ProRoot MTA as detected by ICP-MS. Aged ProRoot MTA samples exhibited a black discolouration and surface calcium carbonate deposition. ProRoot MTA influenced cell counts after direct exposure and its 180-days leachates reduced BMSC viability. After direct BMSC contact with non-aged ProRoot MTA an upregulation of metallothionein (MT1 and MT2A) expression and down-regulation of collagen-1a (Col-1a) and bone sialoprotein (BSP) expression was identified. CONCLUSIONS: Bismuth leaching was observed throughout 180-days observation period from all materials containing bismuth oxide. This negatively influenced cell viability and gene expression associated with bismuth exposure. This is the first study to report that metallothionein gene expression was influenced by exposure to ProRoot MTA.

The relationship between sphingosine-1-phosphate receptor 2 and epidermal growth factor in migration and invasion of oral squamous cell carcinoma.

Sphingosine-1-phosphate (S1P) is a lipid mediator and its binding to the S1P receptor 2 (S1PR2) is reported to regulate cytoskeletal organization. Epidermal growth factor (EGF) has been shown to induce migration and invasion in tumour cells. Since binding of S1P to S1PR2 and EGF to the EGF receptors exhibit some overlapping functionality, this study aimed to determine whether S1PR2 was involved in EGF-induced migration and invasion of oral squamous cell carcinoma (OSCC) lines and to identify any potential crosstalk between the two pathways. Migration was investigated using the scratch wound assay while invasion was studied using the transwell invasion and multicellular tumour spheroid (MCTS) assays. Activity of Rac1, a RhoGTPase, was measured using G-LISA (small GTPase activation assays) while S1P production was indirectly measured via the expression of sphingosine kinase (Sphk). S1PR2 inhibition with 10 µM JTE013 reduced EGF-induced migration, invasion and Rac1 activity, however, stimulation of S1PR2 with 10 µM CYM5478 did not enhance the effect of EGF on migration, invasion or Rac1 activity. The data demonstrated a crosstalk between EGF/EGFR and S1P/S1PR2 pathways at the metabolic level. S1PR2 was not involved in EGF production, but EGF promoted S1P production through the upregulation of Sphk1. In conclusion, OSCC lines could not migrate and invade without S1PR2 regulation, even with EGF stimulation. EGF also activated S1PR2 by stimulating S1P production via Sphk1. The potential for S1PR2 to control cellular motility may lead to promising treatments for OSCC patients and potentially prevent or reduce metastasis.

Comparison of obturation quality in natural and replica teeth root-filled using different sealers and techniques.

OBJECTIVES: This study aims to assess the obturation efficacy of sealers placed with different techniques using microcomputed tomography (µCT) and assess the influence of µCT testing parameters on the obturation data obtained. MATERIALS AND METHODS: Incisors and mesial roots of lower molars with standardized root length were scanned using µCT, and one tooth of each type was 3D printed in acrylic. Two obturation techniques (warm vertical and single cone) and 4 sealer types (AH Plus, BioRoot RCS, Totalfill BC, and Bio-C Sealers) were assessed following storage in Hank's balanced salt solution for 3 and 6 months by assessing gap and void volume percentages on both natural and replica incisor and molar roots. The storage solution was analysed to assess calcium ion leaching. The influence of temperature, tooth positioning, and moisture content of the teeth while µCT scanning was also investigated. RESULTS: The obturation quality in the incisor group was the same using both natural teeth and replicas (p > 0.05). No changes in void volume were identified when comparing the same sealer using different obturation techniques. The premixed sealers used in single-cone obturation exhibited high void volume in the 3D printed replicas in the long term. The temperature, positioning, and moisture content of the teeth did not affect the outcome of µCT testing. CONCLUSIONS: BioRoot RCS, Totalfill BC, and Bio-C Sealers are suitable for obturation of both complex and simple root canal systems using different obturation techniques with BioRoot RCS exhibiting the highest calcium ion release. 3D printed acrylic teeth can be used to assess the obturation quality in uncomplicated root canal systems. µCT parameters had no significant effect on the µCT measurement. CLINICAL RELEVANCE: The single-cone obturation technique with hydraulic sealer is a simple technique that can be used for obturation of all root canal systems.

Novel Chitosan-Silica Hybrid Hydrogels for Cell Encapsulation and Drug Delivery.

Hydrogels constructed from naturally derived polymers provide an aqueous environment that encourages cell growth, however, mechanical properties are poor and degradation can be difficult to predict. Whilst, synthetic hydrogels exhibit some improved mechanical properties, these materials lack biochemical cues for cells growing and have limited biodegradation. To produce hydrogels that support 3D cell cultures to form tissue mimics, materials must exhibit appropriate biological and mechanical properties. In this study, novel organic-inorganic hybrid hydrogels based on chitosan and silica were prepared using the sol-gel technique. The chemical, physical and biological properties of the hydrogels were assessed. Statistical analysis was performed using One-Way ANOVAs and independent-sample t-tests. Fourier transform infrared spectroscopy showed characteristic absorption bands including amide II, Si-O and Si-O-Si confirming formation of hybrid networks. Oscillatory rheometry was used to characterise the sol to gel transition and viscoelastic behaviour of hydrogels. Furthermore, in vitro degradation revealed both chitosan and silica were released over 21 days. The hydrogels exhibited high loading efficiency as total protein loading was released in a week. There were significant differences between TC2G and C2G at all-time points (p < 0.05). The viability of osteoblasts seeded on, and encapsulated within, the hydrogels was >70% over 168 h culture and antimicrobial activity was demonstrated against Pseudomonas aeruginosa and Enterococcus faecalis. The hydrogels developed here offer alternatives for biopolymer hydrogels for biomedical use, including for application in drug/cell delivery and for bone tissue engineering.

In vitro bioactivity of titanium-doped bioglass.

Previous studies have suggested that incorporating relatively small quantities of titanium dioxide into bioactive glasses may result in an increase in bioactivity and hydroxyapatite formation. The present work therefore investigated the in vitro bioactivity of a titanium doped bioglass and compared the results with 45S5 bioglass. Apatite formation was evaluated for bioglass and Ti-bioglass in the presence and absence of foetal calf serum. Scanning electron microscopy (SEM) images were used to evaluate the surface development and energy dispersive X-ray measurements provided information on the elemental ratios. X-ray diffraction spectra confirmed the presence of apatite formation. Cell viability was assessed for bone marrow stromal cells under direct and indirect contact conditions and cell adhesion was assessed using SEM.

Advanced Time-Stepping Interpretation of Fly-Scan Continuous Rotation Synchrotron Tomography of Dental Enamel Demineralization.

High-resolution spatial and temporal analysis and 3D visualization of time-dependent processes, such as human dental enamel acid demineralization, often present a challenging task. Overcoming this challenge often requires the development of special methods. Dental caries remains one of the most important oral diseases that involves the demineralization of hard dental tissues as a consequence of acid production by oral bacteria. Enamel has a hierarchically organized architecture that extends down to the nanostructural level and requires high resolution to study its evolution in detail. Enamel demineralization is a dynamic process that is best investigated with the help of in situ experiments. In previous studies, synchrotron tomography was applied to study the 3D enamel structure at certain time points (time-lapse tomography). Here, another distinct approach to time-evolving tomography studies is presented, whereby the sample image is reconstructed as it undergoes continuous rotation over a virtually unlimited angular range. The resulting (single) data set contains the data for multiple (potentially overlapping) intermediate tomograms that can be extracted and analyzed as desired using time-stepping selection of data subsets from the continuous fly-scan recording. One of the advantages of this approach is that it reduces the amount of time required to collect an equivalent number of single tomograms. Another advantage is that the nominal time step between successive reconstructions can be significantly reduced. We applied this approach to the study of acidic enamel demineralization and observed the progression of demineralization over time steps significantly smaller than the total acquisition time of a single tomogram, with a voxel size smaller than 0.5 µm. It is expected that the approach presented in this paper can be useful for high-resolution studies of other dynamic processes and for assessing small structural modifications in evolving hierarchical materials.

The DIAD Approach to Correlative Synchrotron X-ray Imaging and Diffraction Analysis of Human Enamel.

The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source (Didcot, U.K.) implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales. Namely, the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging (radiography and tomography) and reciprocal space scattering (diffraction). The switching between the two principal modes is made smooth and rapid by design, so that the data collected is interlaced to obtain near-simultaneous multimodal characterization. Different specific photon energies are used for each mode, and the interlacing of acquisition steps allows conducting static and dynamic experiments. Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice, namely, the methods for gauge volume collocation under different modes of beam-sample interaction. To address this challenge, experiments were conducted at DIAD devoted to the study of human dental enamel, a hierarchical structure composed of hydroxyapatite mineral nanocrystals, as a static sample previously affected by dental caries (tooth decay) as well as under dynamic conditions simulating the process of acid demineralization. Collocation and correlation were achieved between WAXS (wide-angle X-ray scattering), 2D (radiographic), and 3D (tomographic) imaging. While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure, X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation (texture). Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities. Diffraction gauge volumes were identified and visualized within the tomographic data sets, revealing the underlying local information to support the interpretation of the diffraction patterns. The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.

Differing responses of osteogenic cell lines to ß-glycerophosphate.

Ascorbic acid (Asc), dexamethasone (Dex) and ß-glycerophosphate (ß-Gly) are commonly used to promote osteogenic behaviour by osteoblasts in vitro. According to the literature, several osteosarcoma cells lines appear to respond differently to the latter with regards to proliferation kinetics and osteogenic gene transcription. Unsurprisingly, these differences lead to contrasting data between publications that necessitate preliminary studies to confirm the phenotype of the chosen osteosarcoma cell line in the presence of Asc, Dex and ß-Gly. The present study exposed Saos-2 cells to different combinations of Asc, Dex and ß-Gly for 14 days and compared the response with immortalised human mesenchymal stromal/stem cells (MSCs). Cell numbers, cytotoxicity, mineralised matrix deposition and cell proliferation were analysed to assess osteoblast-like behaviour in the presence of Asc, Dex and ß-Gly. Additionally, gene expression of runt-related transcription factor 2 (RUNX2); osteocalcin (OCN); alkaline phosphatase (ALP); phosphate regulating endopeptidase homolog X-linked (PHEX); marker of proliferation MKI67 and proliferating cell nuclear antigen (PCNA) was performed every two days during the 14-day cultures. It was found that proliferation of Saos-2 cells was significantly decreased by the presence of ß-Gly which contrasted with hMSCs where no change was observed. Furthermore, unlike hMSCs, Saos-2 cells demonstrated an upregulated expression of late osteoblastic markers, OCN and PHEX that suggested ß-Gly could affect later stages of osteogenic differentiation. In summary, it is important to consider that ß-Gly significantly affects key cell processes of Saos-2 when using it as an osteoblast-like cell model.

Time-Lapse In Situ 3D Imaging Analysis of Human Enamel Demineralisation Using X-ray Synchrotron Tomography.

Caries is a chronic disease that causes the alteration of the structure of dental tissues by acid dissolution (in enamel, dentine and cementum) and proteolytic degradation (dentine and cementum) and generates an important cost of care. There is a need to visualise and characterise the acid dissolution process on enamel due to its hierarchical structure leading to complex structural modifications. The process starts at the enamel surface and progresses into depth, which necessitates the study of the internal enamel structure. Artificial demineralisation is usually employed to simulate the process experimentally. In the present study, the demineralisation of human enamel was studied using surface analysis carried out with atomic force microscopy as well as 3D internal analysis using synchrotron X-ray tomography during acid exposure with repeated scans to generate a time-lapse visualisation sequence. Two-dimensional analysis from projections and virtual slices and 3D analysis of the enamel mass provided details of tissue changes at the level of the rods and inter-rod substance. In addition to the visualisation of structural modifications, the rate of dissolution was determined, which demonstrated the feasibility and usefulness of these techniques. The temporal analysis of enamel demineralisation is not limited to dissolution and can be applied to other experimental conditions for the analysis of treated enamel or remineralisation.

Synchrotron X-ray Studies of the Structural and Functional Hierarchies in Mineralised Human Dental Enamel: A State-of-the-Art Review.

Hard dental tissues possess a complex hierarchical structure that is particularly evident in enamel, the most mineralised substance in the human body. Its complex and interlinked organisation at the Ångstrom (crystal lattice), nano-, micro-, and macro-scales is the result of evolutionary optimisation for mechanical and functional performance: hardness and stiffness, fracture toughness, thermal, and chemical resistance. Understanding the physical-chemical-structural relationships at each scale requires the application of appropriately sensitive and resolving probes. Synchrotron X-ray techniques offer the possibility to progress significantly beyond the capabilities of conventional laboratory instruments, i.e., X-ray diffractometers, and electron and atomic force microscopes. The last few decades have witnessed the accumulation of results obtained from X-ray scattering (diffraction), spectroscopy (including polarisation analysis), and imaging (including ptychography and tomography). The current article presents a multi-disciplinary review of nearly 40 years of discoveries and advancements, primarily pertaining to the study of enamel and its demineralisation (caries), but also linked to the investigations of other mineralised tissues such as dentine, bone, etc. The modelling approaches informed by these observations are also overviewed. The strategic aim of the present review was to identify and evaluate prospective avenues for analysing dental tissues and developing treatments and prophylaxis for improved dental health.

Multi-resolution Correlative Ultrastructural and Chemical Analysis of Carious Enamel by Scanning Microscopy and Tomographic Imaging.

Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.

Alginate hydrogel has a negative impact on in vitro collagen 1 deposition by fibroblasts.

Hydrogels have been widely investigated as 3D culture substrates because of their reported structural similarity to the extracellular matrix (ECM). Limited ECM deposition, however, occurs within these materials, so the resulting "tissues" bear little resemblance to those found in the body. Here matrix deposition by fibroblasts encapsulated within a calcium alginate (Ca-alg) hydrogel was investigated. Although the cells transcribed mRNA for coll Iα over a period of 3 weeks, very little collagen protein deposition was observed within the gel by histology or immunohistochemistry (IHC). Although molecular diffusion demonstrated charge dependency, this did not prevent the flux of both positively and negative charged amino acids through the gel, suggesting that the absence of ECM could not be attributed to substrate limitation. The flux of protein, however, was charge-dependent as proteins with a net negative charge passed quickly through the Ca-alg into the medium. The minimal collagen deposition within the Ca-alg was attributed to a combination of rapid movement of negatively charged procollagen through the gel and steric hindrance of fibril formation.

Low intensity ultrasound stimulates osteoblast migration at different frequencies.

This study investigated the effects of different frequencies of low intensity ultrasound on osteoblast migration using an in vitro scratch-wound healing assay. Mouse calvarial-derived MC3T3-E1 osteoblasts in culture were exposed to continuous 45 kHz ultrasound (25 mW/cm(2)) or pulsed 1 MHz ultrasound (250 mW/cm(2)) for 30 min followed by 2 days' culture. Ultrasound treatment with either kHz or MHz output similarly and significantly increased cell numbers after 2 days in culture compared with untreated control cultures. In the scratch-wound healing assay the presence of the cell proliferation inhibitor mitomycin C (MMC) did not influence scratch-wound closure in control cultures indicating that cell migration was responsible for the in vitro wound healing. Application of ultrasound significantly stimulated wound closure. MMC did not affect kHz-stimulated in vitro wound healing; however, MMC reduced in part the scratch-wound closure rate in MHz-treated cultures suggesting that enhanced cell proliferation as well as migration was involved in the healing promoted by MHz ultrasound. In conclusion, both continuous kHz and pulsed MHz ultrasound promoted osteoblastic migration; however, subtle differences were apparent in the manner the different ultrasound regimens enhanced in vitro scratch-wound healing.

A 3D Printed Device for In Vitro Generation of Stratified Epithelia at the Air-Liquid Interface.

Air-liquid interface (ALI) cultures are used to produce stratified epithelial tissues in vitro, notably for the production of oral mucosal equivalents. Currently, there are few purpose-built devices, which aim to enhance the ease and reproducibility of generating such tissue. Most ALI cultures utilize stainless steel grids or cell culture inserts to elevate the matrix or scaffold to the surface of the culture media. In this study, a novel buoyant epithelial culture device (BECD) was designed to both contain a fibroblast-seeded collagen hydrogel and float in culture media, thereby automatically maintaining the ALI without further user intervention. BECDs aim to mitigate several issues associated with ALI culture; reducing the chance of media flooding the epithelial layer from physical disturbance, reducing technique sensitivity for less-experienced users, and improving the reproducibility of the epithelia generated. H400 oral squamous cell carcinoma cells cultured in BECDs for 7, 14, and 21 days showed continuous increase in epithelial tissue thickness with expected localization of epithelial differentiation markers: cytokeratin 5, involucrin, and E-cadherin. Fused filament fabrication three-dimensional printing with polypropylene used in BECD production allows for rapid turnover and design iteration, presenting a versatile, adaptable, and useful tool for application in in vitro cell culture.

Traditional Multiwell Plates and Petri Dishes Limit the Evaluation of the Effects of Ultrasound on Cells In Vitro.

Ultrasound accelerates healing in fractured bone; however, the mechanisms responsible are poorly understood. Experimental setups and ultrasound exposures vary or are not adequately characterized across studies, resulting in inter-study variation and difficulty in concluding biological effects. This study investigated experimental variability introduced through the cell culture platform used. Continuous wave ultrasound (45 kHz; 10, 25 or 75 mW/cm2, 5 min/d) was applied, using a Duoson device, to Saos-2 cells seeded in multiwell plates or Petri dishes. Pressure field and vibration quantification and finite-element modelling suggested formation of complex interference patterns, resulting in localized displacement and velocity gradients, more pronounced in multiwell plates. Cell experiments revealed lower metabolic activities in both culture platforms at higher ultrasound intensities and absence of mineralization in certain regions of multiwell plates but not in Petri dishes. Thus, the same transducer produced variable results in different cell culture platforms. Analysis on Petri dishes further revealed that higher intensities reduced vinculin expression and distorted cell morphology, while causing mitochondrial and endoplasmic reticulum damage and accumulation of cells in sub-G1 phase, leading to cell death. More defined experimental setups and reproducible ultrasound exposure systems are required to study the real effect of ultrasound on cells for development of effective ultrasound-based therapies not just limited to bone repair and regeneration.

4D microstructural changes in dentinal tubules during acid demineralisation.

OBJECTIVE: Dental erosion is a common oral condition caused by chronic exposure to acids from intrinsic/extrinsic sources. Repeated acid exposure can lead to the irreversible loss of dental hard tissues (enamel, dentine, cementum). Dentine can become exposed to acid following severe enamel erosion, crown fracture, or gingival recession. Causing hypersensitivity, poor aesthetics, and potential pulp involvement. Improving treatments that can restore the structural integrity and aesthetics are therefore highly desirable. Such developments require a good understanding of how acid demineralisation progresses where relatively little is known in terms of intertubular dentine (ITD) and peritubular dentine (PTD) microstructure. To obtain further insight, this study proposes a new in vitro method for performing demineralisation studies of dentine. METHODS: Advanced high-speed synchrotron X-ray microtomography (SXM), with high spatial (0.325 µm) and temporal (15 min) resolution, was used to conduct the first in vitro, time-resolved 3D (4D) study of the microstructural changes in the ITD and PTD phases of human dentine samples (∼0.8 × 0.8 × 5 mm) during 6 h of continuous acid exposure. RESULTS: Different demineralisation rates of ITD (1.79 µm/min) and PTD (1.94 µm/min) and their progressive width-depth profiles were quantified, which provide insight for understanding the mechanisms of dentine demineralisation. SIGNIFICANCE: Insights obtained from morphological characterisations and the demineralisation process of ITD and PTD during acid demineralisation would help understand the demineralisation process and potentially aid in developing new therapeutic dentine treatments. This method enables continuous examination of relatively large volumes of dentine during demineralisation and also demonstrates the potential for studying the remineralisation process of proposed therapeutic dentine treatments.

Acid-induced demineralisation of human enamel as a function of time and pH observed using X-ray and polarised light imaging.

Acid-induced enamel demineralisation affects many individuals either by exposure to acidic diets, acidic gas pollution (dental erosion) or to dental plaque acids (dental caries). This study aimed to develop in situ X-ray and light imaging methods to determine progression of enamel demineralisation and the dynamic relationship between acid pH and mineral density. Hourly digital microradiograph time-lapse sequences showed the depth of enamel demineralisation in 500 µm thick sections progressed with time from the surface towards the dentine following a power-law function, which was 21% faster than the lateral demineralisation progression after exposure for 85 h to lactic acid (10%, pH 2.2). The minimum greyscale remaining (mineral content) within the induced enamel lesion followed an exponential decay, while the accumulated total greyscale loss with time was linear, which showed a constant anisotropic mineral release within the enamel architecture. This 85 h demineralisation method studied by polarised light microscopy time-lapse sequences showed that once the demineralisation front reached the enamel Hunter-Schreger bands, there was preferential demineralisation along those bands. Mineral density loss was linear with increasing pH acidity between pH 5.2 and pH 4.0 (with 0.4 pH increments) when incubated over a 3-week period exposed to 0.5% lactic acid. At pH 4.0, there was complete mineral loss in the centre of the demineralised area after the 3-week period and the linear function intercepted the x-axis at ~ pH 5.5, near the critical pH for hydroxyapatite (HAp). These observations showed how intrinsic enamel structure and pH affected the progression of demineralisation. STATEMENT OF SIGNIFICANCE: Hydroxyapatite crystallites (HAp) in human enamel dissolve when exposed to an acidic environment but little is known about how the intrinsic structures in enamel and pH influence the demineralisation kinetics. We have developed a time-lapse in situ microradiography method to quantify microscopic anisotropic mineral loss dynamics in response to an acid-only caries model. Correlation with polarised light microscopy time-lapse sequences showed that larger structures in enamel also influence demineralisation progression as demineralisation occurred preferentially along the Hunter-Schreger bands (decussating prismatic enamel). The pH-controlled enamel mineral release in a linear manner quantifying the relationship between HAp orientation and acid solubility. These findings should direct the development of improved anti-demineralisation/ remineralisation treatments to retain/ restore the natural intrinsic enamel structure.

Finite Element Modelling and Experimental Validation of the Enamel Demineralisation Process at the Rod Level.

In the past years, a significant amount of effort has been directed at the observation and characterisation of caries using experimental techniques. Nevertheless, relatively little progress has been made in numerical modelling of the underlying demineralisation process. The present study is the first attempt to provide a simplified calculation framework for the numerical simulation of the demineralisation process at the length scale of enamel rods and its validation by comparing the data with statistical analysis of experimental results. FEM model was employed to simulate a time-dependent reaction-diffusion equation process in which H ions diffuse and cause demineralisation of the enamel. The local orientation of the hydroxyapatite crystals was taken into account. Experimental analysis of the demineralising front was performed using advanced high-resolution synchrotron X-ray micro-Computed Tomography. Further experimental investigations were conducted by means of SEM and STEM imaging techniques. Besides establishing and validating the new modelling framework, insights into the role of the etchant solution pH level were obtained. Additionally, some light was shed on the origin of different types of etching patterns by simulating the demineralisation process at different etching angles of attack. The implications of this study pave the way for simulations of enamel demineralisation within different complex scenarios and across the range of length scales. Indeed, the framework proposed can incorporate the presence of chemical species other than H ions and their diffusion and reaction leading to dissolution and re-precipitation of hydroxyapatite. It is the authors' hope and aspiration that ultimately this work will help identify new ways of controlling and preventing caries.

Biofilm viability checker: An open-source tool for automated biofilm viability analysis from confocal microscopy images.

Quantifying biofilm formation on surfaces is challenging because traditional microbiological methods, such as total colony-forming units (CFUs), often rely on manual counting. These are laborious, resource intensive techniques, more susceptible to human error. Confocal laser scanning microscopy (CLSM) is a high-resolution technique that allows 3D visualisation of biofilm architecture. In combination with a live/dead stain, it can be used to quantify biofilm viability on both transparent and opaque surfaces. However, there is little consensus on the appropriate methodology to apply in confocal micrograph processing. In this study, we report the development of an image analysis approach to repeatably quantify biofilm viability and surface coverage. We also demonstrate its use for a range of bacterial species and translational applications. This protocol has been created with ease of use and accessibility in mind, to enable researchers who do not specialise in computational techniques to be confident in applying these methods to analyse biofilm micrographs. Furthermore, the simplicity of the method enables the user to adapt it for their bespoke needs. Validation experiments demonstrate the automated analysis is robust and accurate across a range of bacterial species and an improvement on traditional microbiological analysis. Furthermore, application to translational case studies show the automated method is a reliable measurement of biomass and cell viability. This approach will ensure image analysis is an accessible option for those in the microbiology and biomaterials field, improve current detection approaches and ultimately support the development of novel strategies for preventing biofilm formation by ensuring comparability across studies.

A review of co-culture models to study the oral microenvironment and disease.

Co-cultures allow for the study of cell-cell interactions between different eukaryotic species or with bacteria. Such an approach has enabled researchers to more closely mimic complex tissue structures. This review is focused on co-culture systems modelling the oral cavity, which have been used to evaluate this unique cellular environment and understand disease progression. Over time, these systems have developed significantly from simple 2D eukaryotic cultures and planktonic bacteria to more complex 3D tissue engineered structures and biofilms. Careful selection and design of the co-culture along with critical parameters, such as seeding density and choice of analysis method, have resulted in several advances. This review provides a comparison of existing co-culture systems for the oral environment, with emphasis on progression of 3D models and the opportunity to harness techniques from other fields to improve current methods. While filling a gap in navigating this literature, this review ultimately supports the development of this vital technique in the field of oral biology.