The authors have no acknowledgements.

Collection of the teeth used in this experiment was performed in accordance with the guidelines of the regional Medical Ethics Committee.
1. Specimen Collection and Sample Preparation
<ol>
	<li>Collect 24 sound extracted human (pre)molars from dental practices, and brush with pumice in a low-speed handpiece to a clean (no debris, no gingival remnants) and smooth tooth surface, and finally rinse for 15 s under running tap water.</li>
	<li>Embed the samples to make them fit into the recesses (12 mm x 15 mm x 27.5 mm) of the cylinder of the device.
	<ol>
		<li>Melt the impression compound (one stick of 113 g) on a hotplate (50 &#176;C) for approximately 10 min and dip the occlusal part of the molar in the molten substance, covering the occlusal surface. Place the molar upside down on a microscope slide, and press down until all cup tips touch the glass and wait &#177; 30 s until the impression compound has cooled and set, fixing the tooth.</li>
		<li>Use a syringe to pour 10 mL of polymethylmethacrylate (PMMA) mixture into a silicone mold with inside dimensions of 12 mm x 15 mm x 27.5 mm. Mix PMMA in a fume hood (ratio 13 g polymer: 10 mL monomer) for roughly 25 s using a spatula. Leave to stand for 15 s so that any air bubbles can escape before using.</li>
		<li>Turn the microscope slide upside down and suspend the molar in the silicon mold filled with PMMA. Press the slide down until it touches the mold. The pouring phase lasts approximately 1.5 min at room temperature.</li>
		<li>After setting of the PMMA at room temperature and 1,000 hPa for 20 min, remove the microscope slide and remove the embedded tooth from the silicon mold.</li>
		<li>Measure the total height of the embedded molar and adjust the height to exactly 27.3 mm by incrementally removing the cured PMMA from the bottom with a milling machine equipped with a milling cutter of 16 mm.</li>
	</ol>
	</li>
</ol>
2. Preparing Demineralization Solution
<ol>
	<li>Add 0.1 M lactic acid (50 g), 1.5 mM CaCl2 (1.103 g), 0.9 mM KH2PO4 (0.612 g), 10 mL 1% chloramine, 0.5 ppm F (2.5 mL of 1,000 ppm Fluorid Standard solution) to 4,900 mL of deionized water on a stirring plate.</li>
	<li>Titrate with 10 M KOH (&#177; 50 mL) to pH 4.8 with calibrated pH glass electrode.</li>
</ol>
3. Sample Mounting and Machine Settings of the Rub&#38;Roll (Figure 1)
<ol>
	<li>Remove the cylinder from the container and place 24 specimens in the recesses in the cylinder of the Rub&#38;Roll.</li>
	<li>To adjust loading force, adjust the protrusion of the specimen from the cylinder by placing a shim in the recess underneath the specimen. For no load (0 N) of 8 specimens, use no shim, and for 30 N (8 specimens) and 50 N (8 specimens), use a shim of 1 mm and 1.5 mm, respectively.</li>
	<li>Mount the 2 parts of the cylinder and secure it with a M6 bolt and place the cylinder in the container.</li>
	<li>Fill the container with 500 mL demineralization solution.</li>
	<li>Place the loading rod: a PVC tube (Hardness 73 Shore A) with an outer diameter of 14 mm and an inner diameter 10 mm with an insert of a stainless steel 316 rod (Hardness 130 - 150 HB) with a diameter of 9 mm.</li>
	<li>Set the rotation speed to 20 rpm, to simulate clinical chewing frequency, and let the device run uninterruptedly.</li>
	<li>During the experiment, replace the demineralization solution and PVC tube, and check the pH with a calibrated glass electrode twice a week.</li>
	<li>After 3 months (corresponding to about 1,500,000 cycles) disconnect the cylinder from the container and remove the specimen by disassembling 2 parts of the cylinder, and store the specimen in deionized water. 
	NOTE: All specimens are scanned before and after loading in the Rub&#38;Roll, using a non-contact profilometer.</li>
</ol>
4. Profilometric Scanning, Analysis, and Subtraction
<ol>
	<li>Generate a topographical measurement of the sample using a profilometer.</li>
	<li>Switch on equipment: computer, PSU module, and sensor controller. Place the appropriate sensor and secure with thumbscrew. Then, carefully insert the optical fiber in the sensor controller.</li>
	<li>Select the correct sensor on the sensor controller. The sensor controller will show 4 options (F1 - F4). Press F4 twice and the confocal sensor menu is displayed.
	<ol>
		<li>Press F3 (sensor choice) and scroll to 2 - 10,000 &#181;m. Select (10 mm) and press F4. Press F1 and choose F4 (yes) to save settings to EEPROM. Select LED intensity and turn to position &#34;&#177; 9 o&#39;clock&#34; and press F4 - F2 - F4 taking a &#34;dark reference&#34; of the sensor.</li>
	</ol>
	</li>
	<li>Open the software and choose the option &#34;connect&#34; to the device. Be aware that the measuring table will automatically move to the &#34;home position&#34; search. The home screen appears on the display. Press Tools in the menu bar followed by sensor selection and choose Sensor S29 | 10 - 10,000 &#181;m.
	<ol>
		<li>Press Tools in the menu bar followed by sample rate and choose 300 Hz. Press Tools in the menu bar followed by speed sensor and choose 0 - 100%.</li>
	</ol>
	</li>
	<li>Select Scan in the menu bar. Then select Key Move Stage. Press the yellow area in the middle of the screen to move the measuring table to the center.</li>
	<li>Position the specimen in the center of the measuring table, followed by setting the correct height within the range of the sensor. Place the sensor above the area of interest of the specimen and adjust the distance of the sensor in such a way that the complete sample area to be scanned is located within the focus range of the sensor. The sensor controller indicates whether the height is within the range of the sensor showing a green area in the live data height.</li>
	<li>Choose Settings. Set the average to 2, to ensure each recorded data point is the average of 2 measurements. This will slow down scanning speed, but increase scanning quality. After finishing the settings press OK to return to main scanning setup.</li>
	<li>Position the sensor beam on the upper left corner of the specimen. Set the total scan area to 15 mm x 12 mm with a step size in X and Y direction of 40 &#181;m (0.04 mm), number of steps in X = 375 and Y = 300. Again, smaller steps will increase scan resolution, but also scanning time. Press Scan now to start scanning.</li>
	<li>When the scan is finished after about 10 min, select File in the menu bar, followed by choosing Save As. The scans are standardized in such a way that the scales are always on the same levels.</li>
	<li>Select File in the menu bar, followed by choosing Open file. Select Warpage in the menu bar. Apply a warpage filter of 1 to eliminate the noise of the scanning table and sensor. Select Highest point in the menu bar, and find the highest point on the molar.</li>
	<li>Select Tools in the menu bar, followed by choosing option-scale to the scan configuration. In the scan configuration, set the offset in mm calculated by Z value highest point (measured in 4.10) - 3500. Set the Range in manual, from 0 to 3.6 mm, and press OK.</li>
	<li>Select Load Area in the menu bar to reset the scale. Select File in the menu bar, followed by choosing Save As.</li>
	<li>Subtract scans taken at two different moments in time.
	<ol>
		<li>Select Open in the menu bar in the software. Locate the original scan and the modified scan file in the directory, select files, and press OK. The Options screen will appear, choose in the leveling option: manual leveling option; and in the compensation offset the options respectively, apply to original and apply to modified.</li>
		<li>Select Window in the menu bar, followed by the option create view and finally the option cross section view.</li>
	</ol>
	</li>
	<li>Select the modified surface and move the modified surface over the original surface in horizontal, vertical, and Z direction (height) by holding the control key and press the left and right arrow key for horizontal direction; holding the control key and press up and down arrow key for vertical direction; holding the shift key and press up and down arrow key for Z direction in such a way that the subtracted volume and height shown in the difference view are as low as possible.</li>
	<li>Select File in the menu bar, followed by choosing Save As. Output is read as mean volume loss and mean height loss.</li>
</ol>

<ol>
	<li>Anderson, K., Throckmorton, G. S., Buschang, P. H., Hayasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+bolus+hardness+on+masticatory+kinematics.">The effects of bolus hardness on masticatory kinematics.</a> J Oral Rehabil. 29, 689-696 (2002).</li><li>Koolstra, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+human+masticatory+system.">Dynamics of the human masticatory system.</a> Crit Rev Oral Biol M. 13 (4), 366-376 (2002).</li><li>Lepley, C. R., Throckmorton, G. S., Ceen, R. F., Buschang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+contributions+of+occlusion,+maximum+bite+force,+and+chewing+cycle+kinematics+to+masticatory+performance.">Relative contributions of occlusion, maximum bite force, and chewing cycle kinematics to masticatory performance.</a> Am J Orthod Dentofac. 13, 606-613 (2011).</li><li>Lee, A., He, H., Lyons, K., Swain, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+wear+and+wear+investigations+in+dentistry.">Tooth wear and wear investigations in dentistry.</a> J Oral Rehabil. 39, 217-225 (2012).</li><li>Lussi, A., Megert, B., Shellis, R. P., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+erosive+effect+of+different+dietary+substances+and+medications.">Analysis of the erosive effect of different dietary substances and medications.</a> Br J Nutr. 107, 252-262 (2012).</li><li>Vieira, A., Overweg, E., Ruben, J. L., Huysmans, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toothbrush+abrasion,+simulated+tongue+friction+and+attrition+of+eroded+bovine+enamel+in+vitro.">Toothbrush abrasion, simulated tongue friction and attrition of eroded bovine enamel in vitro.</a> J Dent. 34 (5), 336-342 (2006).</li><li>Heintze, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+qualify+and+validate+wear+simulation+devices+and+methods.">How to qualify and validate wear simulation devices and methods.</a> Dent Mater. 22, 712-734 (2006).</li><li>Lambrechts, P., Dibbles, E., van Landuyt, K., Peumans, M., van Meerbeek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+simulate+wear?+Overview+of+existing+methods.">How to simulate wear? Overview of existing methods.</a> Dent Mater. 22, 693-701 (2006).</li><li>Ruben, J. L., Roeters, F. J. M., Montagner, A. F., Huysmans, M. C. D. N. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+device+to+simulate+oral+ageing:+the+"Rub&amp;Roll".">A multifunctional device to simulate oral ageing: the "Rub&amp;Roll".</a> J Mech Behav Biomed Mater. 30, 75-82 (2014).</li><li>Xu, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+human+masticatory+system+and+masticatory+robotics.">Review of the human masticatory system and masticatory robotics.</a> Mech Mach Theory. 43, 1353-1375 (2008).</li><li>Abrahamsen, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+worn+dentition+pathognomonic+patterns+of+abrasion+and+erosion.">The worn dentition pathognomonic patterns of abrasion and erosion.</a> Int Dent J. 55, 268-276 (2005).</li><li>Khan, F., Young, W. G., Law, V., Priest, J., Daley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cupped+lesions+of+early+onset+dental+erosion+in+young+southeast+Queensland+adults.">Cupped lesions of early onset dental erosion in young southeast Queensland adults.</a> Aust Dent J. 46, 100-107 (2001).</li><li>Montagner, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+Efficacy+and+Fracture+Pattern+of+Adhesives+Submitted+to+Mechanical+Aging+with+the+Rub&amp;Roll+Device.">Bonding Efficacy and Fracture Pattern of Adhesives Submitted to Mechanical Aging with the Rub&amp;Roll Device.</a> J Adhes Dent. 19, 59-68 (2017).</li><li>Martinez Choy, S. E., Lenz, J., Schweizerhof, K., Schmitter, M., Schindler, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Realistic+kinetic+loading+of+the+jaw+system+during+single+chewing+cycles:+a+finite+element+study.">Realistic kinetic loading of the jaw system during single chewing cycles: a finite element study.</a> J Oral Rehabil. 44, 375-384 (2017).</li></ol>

The authors have nothing to disclose.

The application presented here gives a good impression of the clinical relevance of the Rub&#38;Roll. The occlusal morphology that was created in the experiment with the highest loading level is very similar to the clinical presentation of erosive tooth wear (Figure 5)11,12.
The versatility of the set up lies first of all with the solutions used. In the simplest model, water may be used. Loading samples in...

The oral cavity could be considered a harsh environment: humidity, temperature changes due to hot and cold food intake, and mechanical loading with some of the strongest muscles in the human body. Teeth, however, are eminently equipped to withstand these challenges. The enamel is very hard, and the dentin underneath prevents the relatively brittle enamel from fracturing. The mineral component of both materials, hydroxyapatite is of very low solubility and in equilibrium with the supersaturated saliva. Chewing, drinking, and occasional tooth grinding will result in physiological tooth wear during a lifetime1,2,3. Extreme challenges, such as bruxism or habitual chewing on foreign objects, may lead to excessive wear. Recently, the role of erosion in accelerating mechanical tooth wear has been recognized. Tooth erosion has been extensively studied in vitro, but the models used have generally been simple, and mechanical factors have largely been ignored. The clinical interplay between chemical and mechanical wear processes is therefore not fully understood4.
Many in vitro erosion and erosive wear studies have used simple acid immersion of flat polished enamel or dentine samples, using hardness loss or profilometry as the measurement approach5. The introduction of an abrasive component has usually involved tooth brushing action, or sometimes tongue or enamel cusp sliding contacts6. Such studies have shown that enamel erosion results in a softened surface layer, which is easily abraded. Flat surfaces are usually needed because the mechanical loading device cannot handle uneven surfaces, and the measuring techniques for uneven surfaces are also more complicated. However, most erosive tooth wear in adolescents is seen on occlusal cusps, and abrasion by chewing food is expected to be the most relevant mechanical factor in occlusal erosive wear. The ideal oral wear machine that mimics the oral environment in all details does not exist, and most in vitro models will not allow for natural occlusal surfaces of teeth to be either exposed or measured7,8.
Our laboratory recently introduced a novel device, which conforms to many of Heintze&#39;s7 specifications and tolerances of oral wear simulation models, and that enables the user to perform wear and loading studies separately or simultaneously in an erosive and/or abrasive environment. The new device (Rub&#38;Roll) consists of a stirring machine and a container (Figure 1a). In the container, a cylinder with specimens can be mounted. Between the cylinder and the inner wall of the container one of more rods are placed (Figure 1b). By starting the stirring motor, the rod rotates over the specimens in the cylinder (Figure 1c). Using shims, different forces can be applied on the specimens. For a comprehensive description of the design, construction, operation mechanism, and features of the device refer to the paper introducing and discussing the device9. The device is robust, not technically demanding, and can apply loads to 32 specimens simultaneously. The antagonist force is moving over the specimen surface while maintaining smooth, continuous contact, which is comparable to normal chewing10. Here we present an application to model erosive wear of the occlusal surfaces of natural teeth, and we demonstrate the clinical relevance and versatility of the method.

<table><tbody><tr><td>Low speed handpiece</td><td>KAVO, Leutkirch imm Allgau, Germany</td><td></td><td>Dental equipment</td></tr><tr><td>Brush for handpiece</td><td>KAVO, Leutkirch imm Allgau, Germany</td><td></td><td>Dental equipment</td></tr><tr><td>Pumish</td><td></td><td></td><td>Dental equipment</td></tr><tr><td>Human third molars</td><td></td><td></td><td></td></tr><tr><td>Impression compound green</td><td>Kerr, Bioggio, Switzerland</td><td>Art.nr. 00444</td><td></td></tr><tr><td>Microscope slide</td><td>Menzel, Braunschweig, Germany</td><td></td><td>76 x 26 mm</td></tr><tr><td>Autoplast Cold curing denture base material</td><td>Candulor, Wangen, Switzerland</td><td></td><td></td></tr><tr><td>Silicone mold with inside dimensions of 12 x 15 x 27.5</td><td>3M Espe Neuss, Germany</td><td>Express STD</td><td></td></tr><tr><td>Pressure vessel</td><td>Al Dente, Meckenbeuren, Germany</td><td>581-009-024/25</td><td></td></tr><tr><td>Milling cutter &amp;oslash;16mm</td><td>Format, Germany</td><td>HSSCo8 nr. 21691600</td><td></td></tr><tr><td>Milling machine</td><td>Weiss Machine Tools</td><td>WMD 20 LV</td><td></td></tr><tr><td>Rub&amp;amp;Roll</td><td>UMCN , Nijmegen The Netherlands</td><td></td><td>Technical workshop</td></tr><tr><td>Rub&amp;amp;Roll container</td><td>UMCN , Nijmegen The Netherlands</td><td></td><td>Technical workshop</td></tr><tr><td>Rub&amp;amp;Roll cylinder sample holder</td><td>UMCN , Nijmegen The Netherlands</td><td></td><td>Technical workshop</td></tr><tr><td>Rub&amp;amp;Roll motor</td><td>UMCN , Nijmegen The Netherlands</td><td></td><td>Technical workshop</td></tr><tr><td>Shim: Silicone plate massive 1 mm/ 1,5mm, 60 &amp;plusmn; 5&amp;deg; Shore A, red</td><td>Peter van den Berg afdichtingstechniek, Barendrecht</td><td></td><td></td></tr><tr><td>Lactid acid extra pure 88%</td><td>Boom, The Netherlands</td><td>CAS nummer: 79-33-4</td><td></td></tr><tr><td>Calcium Chloride dihydrate CaCL&lt;sub&gt;2&lt;/sub&gt; .2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck, Darmstadt, Germany</td><td>CAS nummer: 10043-52-4</td><td></td></tr><tr><td>Pottassium dihydrogen Phosphate KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck, Darmstadt, Germany</td><td>CAS nummer: 7778-77-0</td><td></td></tr><tr><td>Chloramine T (sodium salt) trihydrate for synthesis CH&lt;sub&gt;3&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;SO&lt;sub&gt;2&lt;/sub&gt;NClNa&amp;middot;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck, Darmstadt, Germany</td><td>CAS nummer: 7080-50-4</td><td></td></tr><tr><td>Natriumfluoride standard solution 1000mg/L F Certipur</td><td>Merck, Darmstadt, Germany</td><td>CAS nummer: 7681-49-4</td><td></td></tr><tr><td>Deionized water</td><td></td><td></td><td></td></tr><tr><td>Kaliumhydroxide, pellets EMSURE analytical reagent KOH</td><td>Merck, Darmstadt, Germany</td><td>CAS nummer: 1310-58-3</td><td></td></tr><tr><td>PVC tube(Hardness73 Shore A)outer diameter 14mm inner diameter 10mm</td><td>DEUTSCH &amp;amp; NEUMANN, Germany</td><td>Art.nr. 3501014</td><td></td></tr><tr><td>Insert of a stainless steel 316 (Hardness 130&amp;ndash;150 HB) diameter 9mm</td><td>UMCN , Nijmegen The Netherlands</td><td></td><td>Technical workshop</td></tr><tr><td>pH glass electrode</td><td>WTW, Weilheim, Germany</td><td>Sentix 61 103640</td><td></td></tr><tr><td>Non contact Profilometer Proscan 2100</td><td>Scantron Industrial Products Ltd, Taunton, UK</td><td></td><td>http://www.scantronltd.co.uk</td></tr><tr><td>Software version Proscan 2100 2.1.1.15A+ Sensor S29 / 10-10000 microns</td><td>Scantron Industrial Products Ltd, Taunton, UK</td><td></td><td></td></tr><tr><td>Software version Proform</td><td>Scantron Industrial Products Ltd, Taunton, UK</td><td></td><td></td></tr><tr><td>Stereomicroscope Leica</td><td>www.leica-microsystems.com</td><td>M50</td><td></td></tr><tr><td>Photocamera Canon</td><td>Canon Japan</td><td>EOS 50D</td><td></td></tr><tr><td>Syringe</td><td>BD Plastipak, Spain</td><td>20 ml.</td><td></td></tr><tr><td>Hotplate</td><td>Schott instruments Mainz</td><td>SLK1</td><td></td></tr><tr><td>Silone impression material (Vinyl Polysiloxane Expres)</td><td>3M Espe , USA</td><td>Regular</td><td></td></tr><tr><td>Stirring Plate</td><td>IKA Werke, Germany</td><td>KMO2 Basic</td><td></td></tr></tbody></table>

mimicking and measuring occlusal erosive tooth wear with the "rub&amp;roll" and non-contact profilometry

Chewing, drinking, and occasional tooth grinding will result in physiological tooth wear during a lifetime. Extreme challenges, such as bruxism or habitual chewing on foreign objects, may lead to excessive wear. Recently, the role of erosion in accelerating mechanical tooth wear has been recognized, but the interplay between chemical and mechanical wear processes has not been extensively studied. Our laboratory recently introduced a novel oral wear simulation device, the Rub&#38;Roll, that enables the user to perform wear and loading studies separately or simultaneously in an erosive and/or abrasive environment. This manuscript describes an application of the device: the combined mechanical and erosive loading of extracted human (pre)molars in a simulated chewing movement, with a controlled application of force, velocity, fluid, and time, and the application of non-contact profilometry in visualizing and measuring the resulting wear pattern. The occlusal morphology that was created in the experiment with the highest loading level is very similar to the clinical presentation of erosive wear.

We exposed human molar teeth (n = 8 per group) to an acidic aqueous solution at pH 4.8 in the Rub&#38;Roll, for 3 months. This corresponds to a clinical functioning time of about 6 years. The mechanical load applied was: no load (0 N), 30 N, or 50 N.
Mean occlusal surface height loss for the three groups was: 76 &#177; 20 &#181;m for 0 N; 161 &#177; 40 &#181;m for 30 N; and 266 &#177; 101 &#181;m for 50 N (F...

Watch this Scientific Journal Video about Mimicking and Measuring Occlusal Erosive Tooth Wear with the "Rub&Roll" and Non-contact Profilometry at JoVE.com

Mimicking and Measuring Occlusal Erosive Tooth Wear with the "Rub&Roll" and Non-contact Profilometry

The Rub&#38;Roll can mimic the chewing cycle, allowing variation of chewing force, sliding distance, chewing velocity, number of cycles, and frequency, and with a combination of erosive and abrasive challenges can result in a complex simulation of oral ageing.

Mimicking and Measuring Occlusal Erosive Tooth Wear with the "Rub&amp;Roll" and Non-contact Profilometry

Chewing, drinking, and occasional tooth grinding will result in physiological tooth wear during a lifetime. Extreme challenges, such as bruxism or ...

mimicking-measuring-occlusal-erosive-tooth-wear-with-rub-roll-non

Research

JoVE Journal

Bioengineering

12.1K Views.  Radboud University Medical Center. De Rub & Roll kunnen nabootsen de kauwen cyclus, waardoor variatie van kauwen kracht, glijden afstand kauwen snelheid, aantal cycli, en frequentie, met een combinatie van erosieve en schurende uitdagingen kan resulteren in een complexe simulatie van mondelinge vergrijzing.

Video: Mimicking and Measuring Occlusal Erosive Tooth Wear with the "Rub&Roll" and Non-contact Profilometry

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Experiments on Ultrasonic Lubrication Using a Piezoelectrically-assisted Tribometer and Optical Profilometer

Friction and wear are detrimental to engineered systems. Ultrasonic lubrication is achieved when the interface between two sliding surfaces is vibrated at a frequency above the acoustic range (20 kHz). As a solid-state technology, ultrasonic lubrication can be used where conventional lubricants are unfeasible or undesirable. Further, ultrasonic lubrication allows for electrical modulation of the effective friction coefficient between two sliding surfaces. This property enables adaptive systems that modify their frictional state and associated dynamic response as the operating conditions change. Surface wear can also be reduced through ultrasonic lubrication. We developed a protocol to investigate the dependence of friction force reduction and wear reduction on the linear sliding velocity between ultrasonically lubricated surfaces. A pin-on-disc tribometer was built which differs from commercial units in that a piezoelectric stack is used to vibrate the pin at 22 kHz normal to the rotating disc surface. Friction and wear metrics including effective friction force, volume loss, and surface roughness are measured without and with ultrasonic vibrations at a constant pressure of 1 to 4 MPa and three different sliding velocities: 20.3, 40.6, and 87 mm/sec. An optical profilometer is utilized to characterize the wear surfaces. The effective friction force is reduced by 62% at 20.3 mm/sec. Consistently with existing theories for ultrasonic lubrication, the percent reduction in friction force diminishes with increasing speed, down to 29% friction force reduction at 87 mm/sec. Wear reduction remains essentially constant (49%) at the three speeds considered.

We present a protocol for using a piezoelectrically-assisted tribometer and optical profilometer to investigate the dependence of ultrasonic wear and friction reduction on linear velocity, contact pressure, and surface properties.

Friction and wear are detrimental to engineered systems. Ultrasonic lubrication is achieved when the interface between two sliding surfaces is vibrated at ...

Engineering

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

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.

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.

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

Behavior

Precision of In Vivo Quantitative Tooth Wear Measurement Using Intra-Oral Scans

Quantitative wear measurement is of increasing interest for measuring tooth wear progression. However, most research on quantitative wear measurement has focused on simulated wear or scanned gypsum casts. A 3D Wear Analysis (3DWA) protocol has been developed that analyzes tooth wear in vivo through intra-oral scanners available to dental clinicians. This study investigated the precision of the 3DWA protocol for measuring wear through maximum height loss (mm) and volume change (mm3). Observational prospective wear data from 55 patients were analyzed after 0-1-, 0-3-, and 0-5-year intervals to determine rates of wear, and convenience samples were chosen to test the protocol&#39;s precision on dentitions scanned twice in one sitting and its intra- and inter-rater precision on scans with 0-3- and 0-5-year intervals. Scans were made using intra-oral scanners (IOS) and superimposed using 3D measurement software. T-tests were performed to determine the structural and random error, and trimmed ranges were calculated to interpret the error. For protocol precision, the mean difference was 0.015 mm (-0.002; 0.032, p = 0.076) for height and -0.111 mm3 (-0.250; 0.023, p = 0.101) for volume. The duplicate measurement error was 0.062 mm for height and 0.268 mm3 for volume. The height measurements were precise enough to measure wear after intervals of 0-3 or 0-5 years; however, volume measurements were susceptible to procedural error and operator sensitivity. The 3DWA protocol is precise enough to adequately measure tooth height loss after intervals of a minimum of 3 years or in patients with severe wear progression, but it is not suited to measuring volumetric changes.

Precision of In Vivo Quantitative Tooth Wear Measurement Using Intra-Oral Scans

Quantitative wear measurement is a method of increasing importance in measuring tooth wear progression. We here describe a protocol, its precision, and its intra/inter-rater precision for the acquisition and superimposition of repeated in vivo scanned dentitions in patients with moderate to severe wear, reporting on both height and volume measurements.

Quantitative wear measurement is of increasing interest for measuring tooth wear progression. However, most research on quantitative wear measurement has ...

Medicine

Quasistatic Load-to-Fracture Test of CAD/CAM Occlusal Veneers on Dentin Analogs

Quasistatic Mechanical Testing for Computer-Aided Design and Manufacturing Occlusal Veneers Cemented to Milled Dentin Analog Material

Under current minimally invasive treatment regimes, minor tooth preparation and thinner biomimetic ceramic restoration are used to preserve the restored tooth&#39;s vitality, aesthetics, and function. New computer-aided design and computer-aided manufacturing (CAD/CAM) ceramic-like material are&#160;now available. To guarantee longevity, a dental clinician must know these&#160;newly launched product&#39;s mechanical strength compared to the relatively brittle glass-matrix ceramic. Furthermore, a tooth substitute has been promoted for laboratory investigation, especially after the pandemic, and more evidentiary support is required for its application.
This study developed a laboratory protocol for a monotonic load-to-fracture test to determine the fracture strength of 1 mm-thick CAD/CAM occlusal veneers. Master dies were milled from high-pressure fiberglass laminate, which has&#160;similar elastic modulus and bond strength as hydrated dentin. They were mounted into polyvinyl chloride (PVC) end caps with cold-curing epoxy resin. Occlusal veneers, also called tabletop restorations, were milled from lithium disilicate (LD) and resin nanoceramic blocks (RNC) and cemented to prepared master dies using dual-cured adhesive resin cement. They were&#160;allowed to cure fully by storing in distilled water for 48 h at 37 &#176;C.
All samples were then placed in a universal testing machine and loaded via a non-fixed 5.5 mm stainless-steel ball that allows lateral movement as would occur against the antagonist&#160;teeth. Compression was applied at a 1 mm/min rate, and the load-displacement graph was generated. The average maximum load-bearing capacity of restorations in the RNC group (3,212.80 &#177; 558.67 N) was significantly higher than in the LD group (2727.10 &#177; 472.41 N) (p &#60; 0.05). No debonding was found during the test.&#160;Both CAD/CAM materials may have a similar flaw distribution. Hertzian cone crack was found at the loading site, whereas radial cracks propagating from the cementation surface were found close to the margin in both groups.

A quasistatic load-to-fracture test with a non-fixed stainless steel ball was developed to determine the fracture strength of minimally invasive posterior computer-aided design and manufacturing restorations cemented to dentin analog materials. This test models the typical loading regime responsible for the fracture of dental restorations.

Under current minimally invasive treatment regimes, minor tooth preparation and thinner biomimetic ceramic restoration are used to preserve the restored ...

Microhardness Protocol for Evaluating Rodent Oral Disease

Microhardness Measurements on Tooth and Alveolar Bone in Rodent Oral Disease Models

The mechanical property, microhardness, is evaluated in dental enamel, dentin, and bone in oral disease models, including dental fluorosis and periodontitis. Micro-CT (&#181;CT) provides 3D imaging information (volume and mineral density) and scanning electron microscopy (SEM) produces microstructure images (enamel prism and bone lacuna-canalicular). Complementarily to structural analysis by &#181;CT and SEM, microhardness is one of the informative parameters to evaluate how structural changes alter mechanical properties. Despite being a useful parameter, studies on microhardness of alveolar bone in oral diseases are limited. To date, divergent microhardness measurement methods have been reported. Since microhardness values vary depending on the sample preparation (polishing and flat surface) and indentation sites, diverse protocols can cause discrepancies among studies. Standardization of the microhardness protocol is essential for consistent and accurate evaluation in oral disease models. In the present study, we demonstrate a standardized protocol for microhardness analysis in tooth and alveolar bone. Specimens used are as follows: for the dental fluorosis model, incisors were collected from mice treated with/without fluoride-containing water for 6 weeks; for ligature-induced periodontal bone resorption (L-PBR) model, alveolar bones with periodontal bone resorption were collected from mice ligated on the maxillary 2nd molar. At 2 weeks after the ligation, the maxilla was collected. Vickers hardness was analyzed in these specimens according to the standardized protocol. The protocol provides detailed materials and methods for resin embedding, serial polishing, and indentation sites for incisors and alveolar. To the best of our knowledge, this is the first standardized microhardness protocol to evaluate the mechanical properties of tooth and alveolar bone in rodent oral disease models.

Author Spotlight: Establishing an Accurate Microhardness Testing Protocol for Craniofacial Tissues

Microhardness is a mechanical property and an informative parameter for evaluating hard tissue pathophysiology. Here, we demonstrate a standardized protocol (sample preparation, polishing, flat surface, and indentation sites) for microhardness analysis in tooth and alveolar bone in rodent oral disease models, namely, dental fluorosis, and ligature-induced periodontal bone resorption.

Mimicking and Measuring Occlusal Erosive Tooth Wear with the "Rub&Roll" and Non-contact Profilometry

Samenvatting

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