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.

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.

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.