I express my gratitude to Prof. Dr. O&#287;uzhan G&#252;nd&#252;z from Marmara University Nanotechnology and Biomaterials Application and Research Center/Marmara University Faculty of Technology Department of Metallurgy and Material Engineering; Prof. Dr. P&#305;nar Y&#305;lmaz Atal&#305; from Marmara University Faculty of Dentistry, Department of Restorative Dentistry; and Dr. Semra &#220;nal Yildirim from Marmara University Genetic and Metabolic Diseases Research and Investigation Center who provided valuable insights and expertise that seriously supported the investigation.

NOTE: This research employed two distinct kinds of flowable dental composite materials: nanohybrid Group P, and nanohybrid Group B manufactured using unique giomer technology. Casarin et al.&#39;s study17 parameters (mean defect depth difference (Ra; &#181;m): 15, standard deviation (&#181;m): 10, alpha error: 0.05, beta error: 0.90) were utilized in a power analysis to estimate sample size.
1. Creation of composite specimens with similar initial surface roughness
<ol>
	<li>Obtain a piece of transparent glass, a rubber gasket, and a piece of transparent tape to make a composite sample with a thickness of 2 mm and a diameter of 7 mm in accordance with ISO specifications18 (Figure 1A).</li>
	<li>After placing the gasket on the transparent tape, apply the composite sample to the gasket, condense it, and close the transparent glass over the gasket and the composite sample (Figure 1B, C).</li>
	<li>Polymerize the composite material using a light curing system for 20 s from the top and 20 s from the bottom (Figure 1D, E). Use the same system for the other sample as well.</li>
	<li>Use a polishing system for the same length of time and, in the same way, to achieve similar levels of roughness on the surfaces of the composite samples to be analyzed (Figure 2A, B). Immerse in distilled water for 24 h after polishing.</li>
	<li>Prepare an additional composite sample from both composite groups and record the initial electron microscope images at different magnifications (1000x, 2000x, 5000x magnification (Figure 3A,B). Coat the samples with gold for 90 s at 18 mA using a sputter coater and examine the samples using scanning electron microscopy (SEM) at an accelerating voltage of 10 kV.</li>
</ol>
2. Stabilization of the samples into acrylic blocks
<ol>
	<li>Find a plastic l-connection hanger element that is used to fix the kitchen terraces to the wall (Figure 4A). The product consists of two parts. The outer part is made of plastic, and the inner part consists of a metal cover. Just use the plastic part.</li>
	<li>Fill the bottom and back of the plastic part with cold-curing pink acrylic and allow the acrylic to polymerize and harden on a flat surface. Subsequently, excise a section of the holder using a diamond cutting disk, and use a monster lab bur to create a groove that will accommodate the fixation of any specimen (Figure 4B).</li>
	<li>Use the silicone impression material to make a copy of the plastic and acrylic mixture holder prototype. Then, make a negative version of the prototype. This will allow to make enough holders to hold all the specimens separately (Figure 4C).</li>
	<li>Use cold-curing acrylic to fill in the negative and then create sufficient holders. Next, use cold-curing acrylic to stabilize the composite specimens and mark the region where the instrument will be applied (Figure 4D).</li>
	<li>Collect the profilometric measurements. Go to the measure condition tab in the menu of the profilometer. Click on the Settings tab and make the relevant numerical settings as follows: &#955;c = 0.8, &#955;s = 2.5, and Opt length = 2. These settings perform the read-out of the surface roughness at over 2 mm with a cut-off value of 0.8 mm at a speed of 0.25 mm/s (Figure 5A).</li>
	<li>Mark 2 mm below the top center of the composite specimen with the help of a caliper (Figure 5B). Adjust the sensitive tip of the profilometer to that point. Then, start the profilometric measurement. Measure each sample&#39;s average roughness (Ra) using a contact profilometer.</li>
	<li>Verify the readings using the designated locations of each specimen (middle top). For each reading, move the device&#39;s needle 2 mm inside the indicated region (Figure 5C, D). Measure the surface properties of each specimen 3x and compute the average before and immediately after the instrumentation operations.</li>
</ol>
3. Creating the setup for the scaler application
<ol>
	<li>Obtain a parallelometer (Figure 6A). Fix the acrylic block on the table of the parallelometer (Figure 6B, C).</li>
	<li>Obtain a rubber pipe clamp with a triphone, which is used for fixing pipes by mounting them to a wall, ceiling, or floor. Use this to attach the handpiece of the device to the holder arm of the parallelometer (Figure 7A). Increase the number of clamp parts and the amount of rubber in accordance with the thickness of the handpiece. Thicken the screw part of the clamp with cold-curing acrylic (Figure 7B) so that it can be inserted into the holding arm of the parallelometer (Figure 7C).</li>
	<li>Apply the ultrasonic scaler, which is designed for supragingival deposit removal, to each composite material for 60 s using this unique setup at an angle of 0&#176;, at maximum power, under water cooling, and with equal force on a specified designated region (Figure 8A, B, C, D).</li>
	<li>Following the scaler application, repeat the profilometric measurements and the scanning electron microscope imaging for each sample (Figure 9A,B).</li>
</ol>

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The author declares no conflict of interest.

Research consistently shows that both sonic and ultrasonic scaling can increase the surface roughness of tooth-colored restorative materials, with ultrasonic scaling having a more detrimental effect8,9. Ultrasonic scaling and air-powder polishing can further increase the roughness of composite resin and restoration margins, and the extent of damage is material-dependent6. The type of restorative material used can also impact the extent of surface damage, with flowable and silorane-based composites showing smoother surfaces and lower bacterial count1. The current study&#39;s findings contribute to the existing body of literature on the effects of ultrasonic scaling on the surface roughness of composite fillings.
A comparative analysis of the two groups in this study, nanohybrid Group P (monomer content: Bis-EMA, TEGDMA; filler content: pre-polymerized filler, barium glass, silica filler) and patented giomer nanohybrid Group B (monomer content: Bis-GMA, TEGDMA; filler content: glass filler, fluoroboroaluminosilicate glass), revealed no significant difference in roughness alteration following scaler application. This finding is noteworthy as it indicates that advancements in composite technology, specifically giomer-based formulations, do not inherently convey resistance to scaler-induced roughness alterations. The lack of statistically significant difference in the degree of roughness added to both composite types might be discussed considering the material&#39;s filler content and particle size. While the technology and monomer content vary, the mechanical impact of scaling could be independent of these factors, affecting only the most superficial layer where similarities between the composites may be more pronounced. The results obtained here suggest that while technologically advanced composite materials may offer several clinical benefits, their resistance to mechanical alterations induced by scaling must be critically evaluated. Dental practitioners should consider the implications of these findings when performing scaling on patients with composite restorations and possibly favor the use of less abrasive instruments or techniques to maintain surface integrity.
In order to conduct a comparison by examining the surface change of any material, it is necessary that the initial surface characteristics of the materials being compared are comparable. The minimum amount of energy required for the polymerization of the composite materials used in the present study was reported as 10 J/cm2 by the manufacturers19. According to the manufacturer&#39;s instructions, the light curing system used in the present investigation delivers between 1 J and 3 J of energy per cm2 per second. Checci et al.20 proved that extended curing duration enhances the mechanical characteristics of the composites. In accordance with the aforementioned facts, the light system was applied for the polymerization of the samples for a period of 20 s from the top and 20 s from the bottom. Flowable composites are distinguished by a reduced amount of filler material and a higher concentration of diluent monomers in the composition. Traditionally, these composites were made by maintaining the tiny particle size of conventional hybrid composites but lowering the amount of filler. This allowed for an increased amount of resin, which in turn decreased the viscosity of the mixture1,21. Nevertheless, the mechanical qualities of these composites, such as flexural strength and wear resistance, have been consistently shown to be worse when compared to traditional composites. Flowable composites have been proposed as suitable filler materials for low-stress applications and scenarios that involve challenging access or need effective penetration. In order to acquire similar initial surface roughness values in the present investigation, the tested flowable composite materials were polymerized and polished in the same manner, and similar surface roughness values were validated by analyzing the surface characteristics obtained by a profilometer. No manufacturer recommendation was available regarding the rpm value and polishing time for the polishing system employed in the present study22. The samples were polished to similar initial roughness values by the same individual using a 4-step polishing system, as illustrated in Figure 2.
The evidence regarding the effects of various ultrasonic devices commonly employed by experts is inconclusive23,24,25,26. According to Yousefimanesh et al.26, when the same forces were applied, piezoelectric scalers produced smoother root surfaces than magnetostrictive scalers. Flemmig et al.24 also reported that a magnetostrictive device was more aggressive on root surfaces than a piezoelectric device. Nevertheless, Busslinger et al.23 discovered that a piezoelectric device produced a coarser root surface than a magnetostrictive device following instrumentation. Singh et al.25 found that magnetostrictive and piezoelectric ultrasonic instruments created equivalent surface roughness levels and demonstrated comparable capabilities for creating a physiologically harmonious surface, which differed from the previous investigations. The inconsistent findings emphasize the need for greater study in this area. A recent study examined the effects of several ultrasonic scalers and polishing devices on the wear and roughness of root surfaces10. As a result, it is demonstrated that the application settings might have a significant impact on the roughness of the root surface. Furthermore, using the piezoelectric ultrasonic scaler at maximum intensity at a 0&#176; angle resulted in the biggest wear and roughness levels on the root surfaces. As a result, comparable application conditions were applied to the samples in the current investigation, where the most significant impact was found.
In surface analysis research, it is of great importance to be able to perform reproducible applications and establish standard exposures for all samples in order to avoid bias. Moreover, the capacity to precisely disclose the examined surface after the application is also extremely beneficial to the findings. Therefore, when conducting such studies, it is necessary to establish a setup to standardize individualized application and evaluation parameters. In terms of ultrasonic scaler application, the methodological factors that might affect the findings can be mentioned as follows: adjusted lateral force, tip angulation, power settings, and consistency of the application site to be examined10,24,26,27,28. The current study demonstrates that several critical methodological parameters can be acquired using a parallelometer, an acrylic block for securing the sample, and a custom holder that allows the handpiece to be applied to the sample at the desired angle with minimal contact force. In this methodological approach, the inability to measure and alter the force applied to the specimen represents a potential limitation of the constructed setup. On the other hand, it should be remembered that ultrasonic instruments should be applied to dental and restorative surfaces only in contact with the surface without any force in order to minimize any surface damage. Also, in the current setup, it is possible to clearly define the application zone, which is crucial for the exact analysis of the results revealed after the application.
The surface texture of dental composites is critical for their longevity and performance, as rougher surfaces may harbor more plaque and bacteria, leading to an increased risk for caries and periodontal disease. It has been shown that dental plaque accumulation is influenced by surface roughness, with a threshold of 0.2 &#181;m below which bacterial adherence is limited2. This is supported by in vivo studies that have found a correlation between surface roughness and plaque accumulation29,30. The present study demonstrates that the application of a piezoelectric ultrasonic scaler can indeed increase the roughness of composite material surfaces to a level that leads to plaque deposition. This effect was consistent across both tested material types.
It is also important to consider the limitations of this study. The in vitro nature of the experiment may not precisely replicate clinical conditions, particularly concerning the scaler&#39;s angle and the presence of saliva, which could modulate the effects seen. Long-term studies in a clinical setting would confirm these findings and provide a clearer understanding of the scaler&#39;s impact over time.
In conclusion, the present study underscores the importance of considering the physical effects of hygiene procedures on dental composites. Although the clinical significance of the observed roughness increase requires further investigation, the potential for increased plaque retention should inform clinical practices, emphasizing the need for careful scaling techniques around composite restorations.

Maintaining oral health is a cornerstone of comprehensive dental care, and the role of hygiene in the prevention and treatment of periodontal diseases is well-established. One tool employed during the hygiene phase is the dental ultrasonic scaler, which is used to remove dental calculus and plaque1. However, while the efficacy of the scaler in cleaning tooth surfaces is critical, its impact on restorative materials is a subject of ongoing research and interest within dental materials science. Surface roughness in particular has been shown to be a contributor to the accumulation and retention of plaque2, highlighting the need for an understanding of how commonly used dental instruments affect restorative materials.
Recent studies have conducted comparative analyses on the roughness effects of piezoelectric dental scalers on teeth or composite filling materials3,4,5. Mittal et al.5 found that root surfaces scaled with a piezoelectric scaler were less rough than those scaled with a magnetostrictive scaler, although the former lost more material and had more noticeable scratches. Arabac&#305; et al.3 examined the influence of tip wear on root surface roughness using piezoelectric ultrasonic scalers and found differences in erosion ratio based on tip wear. Goldstein et al.4 reported that a magnetostrictive ultrasonic scaler had more adverse effects on the surface roughness of resin-based restorative materials compared to a sonic scaler. Recent research has shown that the use of ultrasonic scaling and air polishing can significantly increase the surface roughness of composite filling materials6,7. These findings are important as increased surface roughness can lead to bacterial adhesion and compromise the longevity of dental restorations. Therefore, it is crucial for dental professionals to consider the potential impact of these procedures on the surface roughness of composite filling materials.
This study seeks to expand the body of knowledge by investigating the roughness effect induced by piezoelectric ultrasonic dental scalers on restorative materials, specifically two different flowable composite filling materials. Given the prevalence of composite materials in restorative dentistry and their differentiation in terms of monomer content and technology, such as conventional composites versus giomer-based composites, it is imperative to assess whether the use of ultrasonic scalers differently affects these materials6,8,9,10. Flowable composites are defined by a reduced filler content, which ultimately results in diminished mechanical properties. Consequently, these materials are unsuitable for use in high-stress-bearing locations such as cervical tooth regions11. In recent decades, manufacturers have launched a new generation of flowable materials with increased mechanical and physical qualities. These materials are stated to be appropriate for use in a wide variety of direct anterior and posterior restorations, including those exposed to extreme stress. Consequently, it is of clinical value to examine the mechanical and physical qualities of several commercially available high-strength flowable dental composites12. By meticulously comparing the roughness effect of scalers on two distinct flowable composite filling materials, the study aims to inform clinical practice, ensuring that procedures optimize both oral health outcomes and the longevity and aesthetics of these recent restorative materials. In assessing the impact of dental instruments on various surfaces, standardization of application across all groups is crucial for ensuring the accuracy of the data obtained. Standardizing characteristics such as tip type, angulation, wear, applied force, movement in dental scaler applications, and similar initial surface characteristics would enhance the quality of these investigations3,13,14,15,16. The configurations established for similar investigations mostly include elements that feature a scale to quantify the applied force, an item to provide the requisite weight for the handpiece, and a limb or individual to carry and apply the hygiene equipment. Standardizing the setup for ultrasonic dental scalers enhances consistency, minimizes variability due to different individual parameters, and improves diagnostic accuracy for assessing surface alterations. The setup configuration revealed similar initial surface properties established in this study to reduce discrepancies in individual-specific applications and provide better outcomes. Additionally, it is distinctive regarding the various items utilized. Furthermore, the method is straightforward and can be readily adopted by a wide range of medical practitioners.
This investigation, through a standardized and controlled in vitro approach, strives to delineate the effects of ultrasonic dental scaler application that result in significant roughness, which is crucial for refining dental hygiene protocols and enhancing the sustainable health of restored teeth.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Beautifil Flow Plus</td>
			<td>Shofu</td>
			<td></td>
			<td>United States</td>
		</tr>
		<tr>
			<td>Evo MA10 Scanning Electron Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>EWO Typ 990 Paralellometer</td>
			<td>Kavo</td>
			<td></td>
			<td>Germany</td>
		</tr>
		<tr>
			<td>Finishing Discs</td>
			<td>Bisco</td>
			<td></td>
			<td>United States</td>
		</tr>
		<tr>
			<td>G4 Scaler Tip</td>
			<td>Woodpecker</td>
			<td></td>
			<td>China</td>
		</tr>
		<tr>
			<td>Premise Flowable</td>
			<td>Kerr</td>
			<td></td>
			<td>United States</td>
		</tr>
		<tr>
			<td>SC 7620 model sputter coater</td>
			<td>Quorum Technologies</td>
			<td></td>
			<td>UK</td>
		</tr>
		<tr>
			<td>Surftest SJ-210</td>
			<td>Mitutoyo</td>
			<td></td>
			<td>Japan</td>
		</tr>
		<tr>
			<td>UDS-A-LED Dental Scaler</td>
			<td>Woodpecker</td>
			<td></td>
			<td>China</td>
		</tr>
		<tr>
			<td>Valo LED Cordless Curing Light</td>
			<td>Ultradent</td>
			<td></td>
			<td>United States</td>
		</tr>
		<tr>
			<td>Zetaplus Silicon Impression Material</td>
			<td>Zhermack</td>
			<td></td>
			<td>Italy</td>
		</tr>
	</tbody>
</table>

roughness impact of piezoelectric dental scaler on two distinct flowable composite filling materials

Dental ultrasonic scalers are commonly employed in periodontal treatment; however, their ability to roughen tooth surfaces is a worry since roughness may increase plaque production, a key cause of periodontal disease. This research studied the influence of a piezoelectric ultrasonic scaler on the roughness of two distinct flowable composite filling materials. To do this, 10 disc-shaped samples were generated from each of the two flowable composite materials. After standardized polishing, samples were submerged in water for 24 h before the first surface examination using electron microscopy and profilometry. The ultrasonic scaler was applied to a specified location of each sample for 60 s under water cooling and regulated force. Post-scaler surface parameters were again examined. Following the application of the scaler, both composite materials exhibited a notable increase in surface roughness, as determined by profilometry (p &lt; 0.01). Additionally, the observed surface roughness was also qualitatively visualized with scanning electron microscopy. While initial roughness levels were comparable across the two composites (p = 0.143) after scaler application, no substantial discrepancy in surface texture was noticed between them (p = 0.684). The use of a high-power piezoelectric ultrasonic scaler on routinely used flowable composite restorations might generate considerable surface roughness, possibly leading to increased plaque accumulation. Nevertheless, it might be postulated that nanohybrid flowable composite materials having conventional monomer ingredients may demonstrate comparable surface alterations within the limitations of this experiment.

The statistical analyses were done using statistical analysis software. The Wilcoxon Signed Rank Test was performed to assess changes within the group. The Mann Whitney-U Test was employed to undertake intergroup comparisons. The significance level was determined at p &#60; 0.05.
In the intragroup profilometric comparison of both groups, it was noted that the scaler application resulted in a considerable roughness, which can be qualitatively visualized by electron microscope images (Group P, p = 0.005; Group B, p = 0.007; Table 1). In the intergroup profilometric comparison, analysis of the roughness changes after scaler application to both composite types whose beginning roughness levels were determined to be comparable (p = 0.143) showed no significant difference (p = 0.684; Table 2).
The efficacy of the innovative technique in standardizing the use of dental appliances is well shown by the typical outcomes. The notable rise in surface roughness seen in both flowable composite materials after ultrasonic scaler application underscores the potential effects of such devices on dental restorations. This finding highlights the need for meticulous evaluation of treatment parameters and material choice to reduce iatrogenic harm. The methodology developed in this study has the potential to be applied for further investigation of the effects of a variety of dental instruments on a variety of materials and under a variety of clinical conditions, thereby offering valuable insights to both dental practitioners and researchers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67446/67446fig01.jpg" /> 
Figure 1:&#160;Preparation and polymerization of composite disks. (A)&#160;The&#160;equipment required for composite disk preparation. (B)&#160;The application of the composite sample into the gasket. (C)&#160;The condensation of the composite sample between the transparent tape and the transparent glass. (D) The light curing device. (E) The polymerization of the composite sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67446/67446fig02.jpg" /> 
Figure 2: Polishing application. (A) The disks used for polishing. (B) The polishing disk application on the composite sample. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67446/67446fig03.jpg" /> 
Figure 3: SEM images before scaling application. The images are from (A) Group P, (B) Group B. From left to right, 1000x, 2000x, and 5000x magnification are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67446/67446fig04.jpg" /> 
Figure 4: Stabilization of the samples into acrylic blocks. (A)&#160;An example illustrating theplastic l-connection hanger element. (B) Cold-curing acrylic application into the plastic l-connection hanger element and preparation of the prototype of the specimen holder. (C) Using silicon-based impression material in order to create the negative of the holder prototype. (D) Insertion of the composite specimens on the acrylic holder. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67446/67446fig05.jpg" /> 
Figure 5:&#160;Steps for profilometric measurement. (A)&#160;Profilometer settings, (B)&#160;profilometer tip adjustment, (C)&#160;profilometer tip contact with the specimen, (D) reading screen of the profilometer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67446/67446fig06.jpg" /> 
Figure 6:&#160;Fixation of the acrylic block with the composite specimen. (A) An illustration of the parallelometer used for the special setup. (B) The table of the parallelometer. (C) Exact positioning of the acrylic block. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/67446/67446fig07.jpg" /> 
Figure 7:&#160;Preparation for the stabilization of the handpiece of the scaler. (A) Holder arm of the parallelometer. (B) Modification of a rubber pipe clamp with triphone. (C) Fixation of the scaler handpiece to the holder arm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/67446/67446fig08.jpg" /> 
Figure 8:&#160;Ultrasonic scaler application with equal force to a marked area with the help of a specially prepared apparatus. (A) Frontal view, (B) diagonal view, (C) lateral view of the application, (D) ultrasonic dental&#160;scaler. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/67446/67446fig09.jpg" /> 
Figure 9: SEM images after scaling application. The images are from (A) Group P and (B) Group B. From left to right, 1000x, 2000x, and 5000x magnification are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67446/67446fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GROUPS</td>
			<td>Before &#181;m</td>
			<td>After &#181;m</td>
			<td>P value*</td>
		</tr>
		<tr>
			<td></td>
			<td>(Mean&#177; SD)</td>
			<td>(Mean &#177; SD)</td>
			<td></td>
		</tr>
		<tr>
			<td>Group P (n =10)</td>
			<td>0.16 &#177; 0.17</td>
			<td>0.44 &#177; 0.28</td>
			<td>0.005</td>
		</tr>
		<tr>
			<td>Group B (n =10)</td>
			<td>0.25 &#177; 0.26</td>
			<td>0.49 &#177; 0.26</td>
			<td>0.007</td>
		</tr>
		<tr>
			<td>P value#</td>
			<td>0.143</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Intragroup roughness change. SD: Standard Deviation; * Wilcoxon Signed Rank Test, p &#706; 0.01, # Mann Whitney-U Test, p &#706; 0.05.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Groups</td>
			<td>Roughness change, &#181;m</td>
		</tr>
		<tr>
			<td></td>
			<td>(Mean &#177; SD)</td>
		</tr>
		<tr>
			<td>Group P (n = 10)</td>
			<td>0.27 &#177; 0.29</td>
		</tr>
		<tr>
			<td>Group B (n = 10)</td>
			<td>0.23 &#177; 0.18</td>
		</tr>
		<tr>
			<td>P value*</td>
			<td>0.684</td>
		</tr>
	</tbody>
</table>
Table 2: Evaluation of the roughness change between the groups. SD: Standard Deviation; * Mann Whitney-U Test, p &#706; 0.05.

This methodology allows applying a dental appliance on any specimen at any angle with standardized force and stability. This approach might be extensively used in health sciences to standardize the impacts of dental equipment with hand-holding elements such as micromotors, turbines, and ultrasonic scalers on varied surfaces.

Roughness Impact of Piezoelectric Dental Scaler on Two Distinct Flowable Composite Filling Materials

Dental ultrasonic scalers are commonly employed in periodontal treatment; however, their ability to roughen tooth surfaces is a worry since roughness may ...

roughness-impact-piezoelectric-dental-scaler-on-two-distinct-flowable

Nanyang Technological University

Research

JoVE Journal

Behavior

437 Views.  Marmara University. This methodology allows applying a dental appliance on any specimen at any angle with standardized force and stability. This approach might be extensively used in health sciences to standardize the impacts of dental equipment with hand-holding elements such as micromotors, turbines, and ultrasonic scalers on varied surfaces.

Video: Roughness Impact of Piezoelectric Dental Scaler on Two Distinct Flowable Composite Filling Materials

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.
First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

Holistic Facial Composite Creation and Subsequent Video Line-up Eyewitness Identification Paradigm

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim to a crime may create from memory a holistic facial composite of the culprit with the assistance of a police operator. The aim is that the composite is recognized by someone who believes that they know the culprit. For this paradigm, participants view a culprit actor on video and following a delay, participant-witnesses construct a holistic system facial composite. Controls do not construct a composite. From a series of arrays of computer-generated, but realistic faces, the holistic system construction method primarily requires participant-witnesses to select the facial images most closely meeting their memory of the culprit. Variation between faces in successive arrays is reduced until ideally the final image possesses a close likeness to the culprit. Participant-witness directed tools can also alter facial features, configurations between features and holistic properties (e.g., age, distinctiveness, skin tone), all within a whole face context. The procedure is designed to closely match the holistic manner by which humans&#8217; process faces. On completion, based on their memory of the culprit, ratings of composite-culprit similarity are collected from the participant-witnesses. Similar ratings are collected from culprit-acquaintance assessors, as a marker of composite recognition likelihood. Following a further delay, all participants &#8212; including the controls&#160;&#8212; attempt to identify the culprit in either a culprit-present or culprit-absent video line-up, to replicate circumstances in which the police have located the correct culprit, or an innocent suspect. Data of control and participant-witness line-up outcomes are presented, demonstrating the positive influence of holistic composite construction on identification accuracy. Correlational analyses are conducted to measure the relationship between assessor and participant-witness composite-culprit similarity ratings, delay, identification accuracy, and confidence to examine which factors influence video line-up outcomes.

This applied experimental paradigm replicates circumstances by which an eyewitness to a real crime may create a holistic facial composite of the culprit from memory, and then attempt to identify the culprit from a video line-up containing the culprit or one in which he or she is not present.

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim ...

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods require saccharin/sucrose fading, water deprivation and/or extended training to initiate operant responding in rats. This paper describes a novel and efficient method to quickly initiate operant responding for ethanol that is convenient for experimenters and does not require water deprivation or saccharin/sucrose fading, thus eliminating the potential confound of using sweeteners in ethanol operant self-administration studies. With this method, Wistar rats typically acquire and maintain self-administration of a 20% ethanol solution in less than two weeks of training. Furthermore, blood ethanol concentrations and rewards are positively correlated for a 30 min self-administration session. Moreover, naltrexone, an FDA-approved medication for alcohol dependence that has been shown to suppress ethanol self-administration in rodents, dose-dependently decreases alcohol intake and motivation to consume alcohol for rats self-administering 20% ethanol, thus validating the use of this new method to study the reinforcing properties of alcohol in rats.

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

Operant oral self-administration methods are commonly used to study the reinforcing properties of ethanol in animals. However, the standard methods ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

Reducing State Anxiety Using Working Memory Maintenance

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock. Anxiety is probed throughout the safe and threat blocks using the acoustic startle reflex, which is potentiated under threat (Anxiety-Potentiated Startle (APS)). By conducting the Sternberg WM paradigm during the threat of shock and probing the startle response during either the WM maintenance interval or the intertrial interval, it is possible to determine the effect of WM maintenance on APS.

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working ...

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. Studying how chronic sleep restriction during development can affect adult behavior may add to our understanding of the emergence of behavioral symptoms of neurodevelopmental disorders. While there are many methods that can be used to restrict sleep in rodents including forced locomotion, constant disruption, presentation of an aversive stimulus, or electric shock, many of these methods are very stressful and cannot be used in neonatal mice. Here, we describe gentle handling, a sleep deprivation technique that can be used chronically throughout development and into adulthood to achieve sleep restriction. Gentle handling involves close observation of the mice throughout the sleep deprivation period and requires the researcher to gently prod the animals whenever they are inactive or display behaviors associated with sleep. Coupled with EEG recordings, gentle handling could be used to selectively disrupt a specific phase of sleep such as rapid eye movement (REM) sleep. The technique of gentle handling is a powerful tool for the study of the effects of chronic sleep restriction even in neonatal mice that circumvents many of the more stressful procedures used for sleep deprivation.

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. ...

A Two-interval Forced-choice Task for Multisensory Comparisons

We provide a procedure for a psychophysics experiment in humans based on a previously described paradigm aimed to characterize the perceptual duration of intervals within the range of milliseconds of visual, acoustic, and audiovisual aperiodic trains of six pulses. In this task, each of the trials consists of two consecutive intramodal intervals where the participants press the upward arrow key to report that the second stimulus lasted longer than the reference, or the downward arrow key to indicate otherwise. The analysis of the behavior results in psychometric functions of the probability of estimating the comparison stimulus to be longer than the reference, as a function of the comparison intervals. In conclusion, we advance a way of implementing standard programming software to create visual, acoustic, and audiovisual stimuli, and to generate a two-interval forced-choice (2IFC) task by delivering stimuli through noise-blocking headphones and a computer&#39;s monitor.

Psychophysics is essential for studying perception phenomena through sensory information. Here we present a protocol to perform a two-interval forced-choice task as implemented in a previous report on human psychophysics where participants estimated the duration of visual, auditory, or audiovisual intervals of aperiodic trains of pulses.

We provide a procedure for a psychophysics experiment in humans based on a previously described paradigm aimed to characterize the perceptual duration of ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...