Ultrasonic irrigation flows in root canals: effects of ultrasound power and file insertion depth.

Ultrasonic irrigation during root canal treatment can enhance biofilm disruption. The challenge is to improve the fluid flow so that the irrigant reaches areas inaccessible to hand instrumentation. The aim of this study is to experimentally investigate how the flow field and hydrodynamic forces induced by ultrasonic irrigation are influenced by the ultrasound power and file insertion depth. A root canal phantom was 3D printed and used as a mold for the fabrication of a PDMS channel. An ultrasonic instrument with a #15K-file provided the irrigation. The flow field was studied by means of Particle Image Velocimetry (PIV). The time averaged velocity and shear stress distributions were found to vary significantly with ultrasound power. Their maximum values increase sharply for low powers and up to a critical power level. At and above this setting, the flow pattern changes, from the high velocity and shear stress region confined in the vicinity of the tip, to one covering the whole root canal domain. Exceeding this threshold also induces a moderate increase in the maximum velocities and shear stresses. The insertion depth was found to have a smaller effect on the measured velocity and shear stresses. Due to the oscillating nature of the flow, instantaneous maximum velocities and shear stresses can reach much higher values than the mean, especially for high powers. Ultrasonic irrigation will benefit from using a higher power setting as this does produce greater shear stresses near the walls of the root canal leading to the potential for increased biofilm removal.

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.

Maize resistance to witchweed through changes in strigolactone biosynthesis.

Maize (Zea mays) is a major staple crop in Africa, where its yield and the livelihood of millions are compromised by the parasitic witchweed Striga. Germination of Striga is induced by strigolactones exuded from maize roots into the rhizosphere. In a maize germplasm collection, we identified two strigolactones, zealactol and zealactonoic acid, which stimulate less Striga germination than the major maize strigolactone, zealactone. We then showed that a single cytochrome P450, ZmCYP706C37, catalyzes a series of oxidative steps in the maize-strigolactone biosynthetic pathway. Reduction in activity of this enzyme and two others involved in the pathway, ZmMAX1b and ZmCLAMT1, can change strigolactone composition and reduce Striga germination and infection. These results offer prospects for breeding Striga-resistant maize.

Exploring the neurogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins ßIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.

Cavitation in a periodontal pocket by an ultrasonic dental scaler: A numerical investigation.

Periodontal pockets are spaces or holes surrounded by teeth under the gum line. These pockets can become filled with infection-causing bacteria resulting in tissue, bone, and tooth loss. Cavitation produced by the oscillating tip of dental ultrasonic scalers plays a significant role in routine periodontal therapy to clean these areas. Numerical studies were conducted for a scaler vibrating in a periodontal pocket which was simplified to a hole, using ABAQUS based on the finite element method. The simulations consider the three-dimensional, nonlinear, and transient interaction between the vibration and deformation of the scaler tip, the water flow around the scaler and the cavitation formation. The numerical model was validated by comparing results with experimental data for a scaler vibrating in an unbounded liquid, the displacement at the free end of the scaler and the cavitation pattern near the scaler tip displaying excellent agreement. A parametric study for a scaler vibrating in a hole has been carried out in terms of the volume of the hole, the taper ratio (the radius ratio between the circular opening and bottom of the hole), and the immersion depth of the scaler tip in the hole. The amount of cavitation generated is evaluated by the cavitation density (or the void fraction) which is the ratio of the volume of the cavitation occupied in the hole to the total volume of the hole. Numerical results indicate that the cavitation density in the hole increases with the decreasing hole volume and the increasing taper ratio. It is inferred that cleaning effects could be increased if some modifications to the scaler design could be made to increase the blocking effect of the hole during the cleaning process. Cavitation is observed in the hole even if the scaler is placed above the hole and increases with the immersion depth.

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.

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.

Improved biofilm removal using cavitation from a dental ultrasonic scaler vibrating in carbonated water.

The use of cavitation for improving biofilm cleaning is of great interest. There is no system at present that removes the biofilm from medical implants effectively and specifically from dental implants. Cavitation generated by a vibrating dental ultrasonic scaler tip can clean biomaterials such as dental implants. However, the cleaning process must be significantly accelerated for clinical applications. In this study we investigated whether the cavitation could be increased, by operating the scaler in carbonated water with different CO2 concentrations. The cavitation around an ultrasonic scaler tip was recorded with high speed imaging. Image analysis was used to calculate the area of cavitation. Bacterial biofilm was grown on surfaces and its removal was imaged with a high speed camera using the ultrasonic scaler in still and carbonated water. Cavitation increases significantly with increasing carbonation. Cavitation also started earlier around the tips when they were in carbonated water compared to non-carbonated water. Significantly more biofilm was removed when the scaler was operated in carbonated water. Our results suggest that using carbonated water could significantly increase and accelerate cavitation around ultrasonic scalers in a clinical situation and thus improve biofilm removal from dental implants and other biomaterials.

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis.

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

The effect of standoff distance and surface roughness on biofilm disruption using cavitation.

Effective biofilm removal from surfaces in the mouth is a clinical challenge. Cavitation bubbles generated around a dental ultrasonic scaler are being investigated as a method to remove biofilms effectively. It is not known how parameters such as surface roughness and instrument distance from biofilm affect the removal. We grew Strepotococcus sanguinis biofilms on coverslips and titanium discs with varying surface roughness (between 0.02-3.15 µm). Experimental studies were carried out for the biofilm removal using high speed imaging and image analysis to calculate the area of biofilm removed at varying ultrasonic scaler standoff distances from the biofilm. We found that surface roughness up to 2 µm does not adversely affect biofilm removal but a surface roughness of 3 µm caused less biofilm removal. The standoff distance also has different effects depending on the surface roughness but overall a distance of 1 mm is just as effective as a distance of 0.5 mm. The results show significant biofilm removal due to an ultrasonic scaler tip operating for only 2s versus 15-60s in previous studies. The technique developed for high speed imaging and image analysis of biofilm removal can be used to investigate physical biofilm disruption from biomaterial surfaces in other fields.

How does ultrasonic cavitation remove dental bacterial biofilm?

Bacterial biofilm accumulation is problematic in many areas, leading to biofouling in the marine environment and the food industry, and infections in healthcare. Physical disruption of biofilms has become an important area of research. In dentistry, biofilm removal is essential to maintain health. The aim of this study is to observe biofilm disruption due to cavitation generated by a dental ultrasonic scaler (P5XS, Acteon) using a high speed camera and determine how this is achieved. Streptococcus sanguinis biofilm was grown on Thermanox™ coverslips (Nunc, USA) for 4 days. After fixing and staining with crystal violet, biofilm removal was imaged using a high speed camera (AX200, Photron). An ultrasonic scaler tip (tip 10P) was held 2 mm away from the biofilm and operated for 2 s. Bubble oscillations were observed from high speed image sequences and image analysis was used to track bubble motion and calculate changes in bubble radius and velocity on the surface. The results demonstrate that most of the biofilm disruption occurs through cavitation bubbles contacting the surface within 2 s, whether individually or in cavitation clouds. Cleaning occurs through shape oscillating microbubbles on the surface as well as through fluid flow.

High speed imaging of biofilm removal from a dental implant model using ultrasonic cavitation.

OBJECTIVES: Current instruments cannot clean in between dental implant threads and effectively remove biofilm from the rough implant surface without damaging it. Cavitation bubbles have the potential to disrupt biofilms. The aim of this study was to see how biofilms can be disrupted using non-contact cavitation from an ultrasonic scaler, imaged inside a restricted implant pocket model using high speed imaging. METHODS: Streptococcus sanguinis biofilm was grown for 7 days on dental implants. The implants were placed inside a custom made restricted pocket model and immersed inside a water tank. An ultrasonic scaler tip was placed 0.5mm away from the implant surface and operated at medium power or high power for 2s. The biofilm removal process was imaged using a high speed camera operating at 500 fps. Image analysis was used to calculate the amount of biofilm removed from the high speed images. Scanning electron microscopy was done to visualize the implant surface after cleaning. RESULTS: Cavitation was able to remove biofilm from dental implants. More biofilm was removed at high power. Scanning electron microscopy showed that the implant surface was clean at the points where the cavitation was most intense. High speed imaging showed biofilm removal underneath implant threads, in areas next to the ultrasonic scaler tip. SIGNIFICANCE: A high speed imaging protocol has been developed to visualize and quantify biofilm removal from dental implants in vitro. Cavitation bubbles from dental ultrasonic scalers are able to successfully disrupt biofilm in between implant threads.

Numerical investigation of cavitation generated by an ultrasonic dental scaler tip vibrating in a compressible liquid.

Bacterial biofilm accumulation around dental implants is a significant problem leading to peri-implant diseases and implant failure. Cavitation occurring in the cooling water around ultrasonic scaler tips can be used as a novel solution to remove debris without any surface damage. However, current clinically available instruments provide insufficient cavitation around the activated tip surface. To solve this problem a critical understanding of the vibro-acoustic behaviour of the scaler tip and the associated cavitation dynamics is necessary. In this research, we carried out a numerical study for an ultrasound dental scaler with a curved shape tip vibrating in water, using ABAQUS based on the finite element method. We simulated the three-dimensional, nonlinear and transient interaction between the vibration and deformation of the scaler tip, the water flow around the scaler and the cavitation formation and dynamics. The numerical model was well validated with the experiments and there was excellent agreement for displacement at the free end of the scaler. A systematic parametric study has been carried out for the cavitation volume around the scaler tip in terms of the frequency, amplitude and power of the tip vibration. The numerical results indicate that the amount of cavitation around the scaler tip increases with the frequency and amplitude of the vibration. However, if the frequency is far from the natural frequency, the cavitation volume around the free end decreases due to reduced free end vibration amplitude.

Which Parameters Affect Biofilm Removal with Acoustic Cavitation? A Review.

Bacterial biofilms are a cause of contamination in a wide range of medical and biological areas. Ultrasound is a mechanical energy that can remove these biofilms using cavitation and acoustic streaming, which generate shear forces to disrupt biofilm from a surface. The aim of this narrative review is to investigate the literature on the mechanical removal of biofilm using acoustic cavitation to identify the different operating parameters affecting its removal using this method. The properties of the liquid and the properties of the ultrasound have a large impact on the type of cavitation generated. These include gas content, temperature, surface tension, frequency of ultrasound and acoustic pressure. For many of these parameters, more research is required to understand their mechanisms in the area of ultrasonic biofilm removal, and further research will help to optimise this method for effective removal of biofilms from different surfaces.

Impact of scientific and technological advances.

Advancements in research and technology are transforming our world. The dental profession is changing too, in the light of scientific discoveries that are advancing biological technology-from new biomaterials to unravelling the genetic make-up of the human being. As health professionals, we embrace a model of continuous quality improvement and lifelong learning. Our pedagogical approach to incorporating the plethora of scientific-technological advancements calls for us to shift our paradigm from emphasis on skill acquisition to knowledge application. The 2017 ADEE/ADEA workshop provided a forum to explore and discuss strategies to ensure faculty, students and, ultimately, patients are best positioned to exploit the opportunities that arise from integrating new technological advances and research outcomes. Participants discussed methods of incorporating the impact of new technologies and research findings into the education of our dental students. This report serves as a signpost of the way forward and how to promote incorporation of research and technology advances and lifelong learning into the dental education curriculum.

Cleaning lateral morphological features of the root canal: the role of streaming and cavitation.

AIM: To investigate the effects of ultrasonic activation file type, lateral canal location and irrigant on the removal of a biofilm-mimicking hydrogel from a fabricated lateral canal. Additionally, the amount of cavitation and streaming was quantified for these parameters. METHODOLOGY: An intracanal sonochemical dosimetry method was used to quantify the cavitation generated by an IrriSafe 25 mm length, size 25 file inside a root canal model filled with filtered degassed/saturated water or three different concentrations of NaOCl. Removal of a hydrogel, demonstrated previously to be an appropriate biofilm mimic, was recorded to measure the lateral canal cleaning rate from two different instruments (IrriSafe 25 mm length, size 25 and K 21 mm length, size 15) activated with a P5 Suprasson (Satelec) at power P8.5 in degassed/saturated water or NaOCl. Removal rates were compared for significant differences using nonparametric Kruskal-Wallis and/or Mann-Whitney U-tests. Streaming was measured using high-speed particle imaging velocimetry at 250 kfps, analysing both the oscillatory and steady flow inside the lateral canals. RESULTS: There was no significant difference in amount of cavitation between tap water and oversaturated water (P = 0.538), although more cavitation was observed than in degassed water. The highest cavitation signal was generated with NaOCl solutions (1.0%, 4.5%, 9.0%) (P < 0.007) and increased with concentration (P < 0.014). The IrriSafe file outperformed significantly the K-file in removing hydrogel (P < 0.05). Up to 64% of the total hydrogel volume was removed after 20 s. The IrriSafe file typically outperformed the K-file in generating streaming. The oscillatory velocities were higher inside the lateral canal 3 mm compared to 6 mm from WL and were higher for NaOCl than for saturated water, which in turn was higher than for degassed water. CONCLUSIONS: Measurements of cavitation and acoustic streaming have provided insight into their contribution to cleaning. Significant differences in cleaning, cavitation and streaming were found depending on the file type and size, lateral canal location and irrigant used. In general, the IrriSafe file outperformed the K-file, and NaOCl performed better than the other irrigants tested. The cavitation and streaming measurements revealed that both contributed to hydrogel removal and both play a significant role in root canal cleaning.