Name: force system with vertical v-bends: a 3d in vitro assessment of elastic and rigid rectangular archwires
Uploaded: 2018-07-24T12:30:00
Description: Watch this Scientific Journal Video about Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires at JoVE.com

The authors would like to acknowledge all colleagues who made this work possible, especially Drs. Aditya Chhibber and Ravindra Nanda. The authors would like to thank the Biodynamics &#38; Bioengineering Lab at UCONN Health for the facilities provided during the development of this project.

1. Experimental Setup
<ol>
	<li>Mark the precise position for the placement of molar tubes and incisor brackets on the aluminum pegs of the OWT by using a customized &#39;jig&#39;.</li>
	<li>Bond standard self-ligating brackets with composite material. Light cure for 40 seconds.</li>
	<li>Insert a 0.021 x 0.025-inch stainless steel (SS) &#39;ovoid&#39; maxillary archwire into the bracket slots.</li>
	<li>Place the testing apparatus in the glass chamber.</li>
	<li>Check for any unintended archwire activation. Any activa...

<ol>
	<li>Burstone, C. J., Koenig, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+systems+from+an+ideal+arch.">Force systems from an ideal arch.</a> Am J Orthod. 65 (3), 270-289 (1974).</li><li>Koenig, H. A., Burstone, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+systems+from+an+ideal+arch:+Large+deflection+considerations.">Force systems from an ideal arch: Large deflection considerations.</a> Angle Orthod. 59 (1), 11-16 (1989).</li><li>Burstone, C. J., Koenig, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creative+wire+bending:+The+force+system+from+step+and+V+bends.">Creative wire bending: The force system from step and V bends.</a> Am J Orthod and Dentofac Orthop. 93 (1), 59-67 (1988).</li><li>Ronay, F., Kleinert, W., Melsen, B., Burstone, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+system+developed+by+V+bends+in+an+elastic+orthodontic+wire.">Force system developed by V bends in an elastic orthodontic wire.</a> Am J Orthod and Dentofac Orthop. 96 (4), 295-301 (1989).</li><li>Demange, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+situations+in+bend+force+systems.">Equilibrium situations in bend force systems.</a> Am J Orthod and Dentofac Orthop. 98 (4), 333-339 (1990).</li><li>Isaacson, R. J., Lindauer, S. J., Conley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+3-dimensional+arch+wires+to+vertical+V+bends:+Comparisons+with+existing+2-dimensional+data+in+the+lateral+view.">Responses of 3-dimensional arch wires to vertical V bends: Comparisons with existing 2-dimensional data in the lateral view.</a> Semin Orthod. 1 (1), 57-63 (1995).</li><li>Upadhyay, M., Shah, R., Peterson, D., Takafumi, A., Yadav, S., Agarwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+system+generated+by+elastic+archwires+with+vertical+V+bends:+A+three-dimensional+analysis.">Force system generated by elastic archwires with vertical V bends: A three-dimensional analysis.</a> Eur J Orthod. 39 (2), 202-208 (2017).</li><li>Gurgel, J. A., Kerr, S., Powers, J. M., LeCrone, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-deflection+properties+of+superelastic+nickel-titanium+archwires.">Force-deflection properties of superelastic nickel-titanium archwires.</a> Am J Orthod Dentofacial Orthop. 120 (4), 378-382 (2001).</li><li>Gurgel, J. A., Kerr, S., Powers, J. M., Pinzan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torsional+properties+of+commercial+nickel-titanium+wires+during+activation+and+deactivation.">Torsional properties of commercial nickel-titanium wires during activation and deactivation.</a> Am J Orthod Dentofacial Orthop. 120 (1), 76-79 (2001).</li><li>Hazel, R. J., Rohan, G. J., West, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+relaxation+in+orthodontic+arch+wires.">Force relaxation in orthodontic arch wires.</a> Am J Orthod. 86 (5), 396-402 (1984).</li><li>Lundgren, D., Owman-Moll, P., Kurol, J., Martensson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+orthodontic+force+and+tooth+movement+measurements.">Accuracy of orthodontic force and tooth movement measurements.</a> Br J Orthod. 23 (3), 241-248 (1996).</li><li>Goldberg, A. J., Burstone, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+beta+titanium+alloys+for+use+in+orthodontic+appliances.">An evaluation of beta titanium alloys for use in orthodontic appliances.</a> J Dent Res. 58 (2), 593-600 (1979).</li><li>Kusy, R. P., Whitley, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+and+mechanical+characteristics+of+stainless+steel,+titanium-molybdenum,+and+nickel+titanium+archwires.">Thermal and mechanical characteristics of stainless steel, titanium-molybdenum, and nickel titanium archwires.</a> Am J Orthod Dentofacial Orthop. 131 (2), 229-237 (2007).</li><li>Kapila, S., Sachdeva, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+and+clinical+applications+of+orthodontic+wires.">Mechanical properties and clinical applications of orthodontic wires.</a> Am J Orthod Dentofacial Orthop. 96 (2), 100-109 (1989).</li><li>Verstrynge, A., Humbeeck, J. V., Willems, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+evaluation+of+the+material+characteristics+of+stainless+steel+and+beta-titanium+orthodontic+wires.">In-vitro evaluation of the material characteristics of stainless steel and beta-titanium orthodontic wires.</a> Am J Orthod Dentofacial Orthop. 130 (4), 460-470 (2006).</li><li>Tominaga, J. Y., Tanaka, M., Koga, Y., Gonzales, C., Kobayashi, M., Yoshida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+loading+conditions+for+controlled+movement+of+anterior+teeth+in+sliding+mechanics.">Optimal loading conditions for controlled movement of anterior teeth in sliding mechanics.</a> Angle. 79 (6), 1102-1107 (2009).</li><li>Cattaneo, P. M., Dalstra, M., Melsen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+finite+element+method:+A+tool+to+study+orthodontic+tooth+movement.">The finite element method: A tool to study orthodontic tooth movement.</a> J Dent Res. 84 (5), 428-433 (2005).</li><li>Fotos, P. G., Spyrakos, C. C., Bernard, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthodontic+forces+generated+by+a+simulated+archwire+appliance+evaluated+by+the+finite+element+method.">Orthodontic forces generated by a simulated archwire appliance evaluated by the finite element method.</a> Angle Orthod. 60 (4), 277-282 (1990).</li><li>Geramy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+bone+resorption+and+the+center+of+resistance+modification+(3-D+analysis+by+means+of+the+finite+element+method.">Alveolar bone resorption and the center of resistance modification (3-D analysis by means of the finite element method.</a> Am J Orthod Dentofacial Orthop. 117 (4), 399-405 (2000).</li></ol>

Orthodontic archwires have been studied in various ways8,9,10,11. They have also been evaluated for various mechanical properties, but they have seldom been analyzed for determining the force system they are going to create12,13,14,15. Three-point bending tests are po...

An important aspect of clinical orthodontic treatment is the knowledge of the force system produced by multibracket appliances. A clear understanding of the underlying biomechanical principles can help deliver predictable results and minimize potential side effects1. Recent years have seen a trend away from placing bends in archwires by building more activation with bracket position and design; however, comprehensive orthodontic treatment still requires placement of bends in archwires. Bends, when placed in different types and sizes of archwires, can create a wide variety of force systems suitable for different types of tooth movement. Although the force systems can become quite complex when multiple teeth are considered, a helpful starting point can involve a simple two-bracket system.
To date, V-bend mechanics have primarily been analyzed in the second order only, utilizing mathematical models1,2,3,4,5 and/or computer-based analysis/simulations6. This has yielded a basic understanding of the force system involved in the second order interaction of the arch wires with adjacent brackets (Figure 1). However, these methods impose certain boundary conditions in order to run&#160;simulations that might not hold true in actual clinical situations and deviations might occur. Recently, a new in vitro model involving force transducers was proposed for measuring three dimensional (3D) forces and moments created by evaluating not only second order archwire-bracket interactions but also in the third order7. However, the effect of different types of archwires on the force system at various bend positions along the incisor molar archwire span was not evaluated. Also, the study only involved evaluation of elastic orthodontic archwires, which are not the primary archwires on which tooth movement occurs. Therefore, the aim of this study was to evaluate the force system created by the placement of a V-bend at different locations in rectangular stainless steel and beta-titanium archwires in a 3D set up involving the molar and incisor brackets. Clinicians need to know the force system applied on the dentition when a specific combination of archwire bracket combination is used to fix a malocclusion.
The described technique has been developed to study the orthodontic force system in all the three planes of space, mimicking clinical reality. It is to be understood that it is extremely difficult to measure the force system clinically; therefore, such measurements have to be carried out&#160;in vitro. It is assumed that the force system created by a V-bend in the laboratory would be similar if replicated in the patient&#39;s mouth. A workflow was created to evaluate how the experimental set up has to be configured (Figure 2).
The orthodontic wire tester (OWT) is an innovative product developed by Division of Orthodontics in collaboration with the Bioengineering &#38; Biodynamics Laboratory, UConn Health, Farmington, CT, USA (Figure 3). It is designed to accurately mimic the arrangement of the maxillary teeth within the mouth and some intra-oral conditions while providing measurements of the force system created in all the three planes of space. The major mechanical components of the OWT are a Data acquisition device (DAQ), nano Force/Torque Sensors, humidity sensors, temperature sensors, and a personal computer. The testing apparatus is placed in a glass enclosure having temperature/humidity controls. This allows for partial simulation of the intraoral environment. The DAQ serves as the interface for the three sensors:&#160;humidity sensor, force/moment sensor,&#160;thermistor&#160;and the testing apparatus with the sensors situated on a platform&#160;(Figure 3). These are linked to a software program. The software is a platform and a development environment for visual programming and is used to control different types of hardware. It was chosen to automate the orthodontic wire tester.
A series of aluminum pegs are arranged on the testing apparatus to represent the teeth of the maxillary dental arch. Two of the pegs representing the right central incisor and right first molar are connected to sensors/load cells (S1 and S2). A load cell is a mechanical device that can measure the forces and moments applied to it in all the three planes (x-y-z): Fx, Fy, and Fz; and Mx, My, and Mz. The pegs are systematically positioned to create a dental arch form. Each peg is separated from the other by a precisely recorded measurement that is calculated using average tooth widths as observed in patients undergoing orthodontic treatment. The shape chosen for the experiment is an &#39;ovoid&#39; arch form created from a standardized template.

<table border="0" cellpadding="0" cellspacing="0" style="width:1011px;" width="1011">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Force/Torque&#160; Sensors/Transducers</td>
			<td style="width:488px;">Nano17 F/T Sensors,&#160; ATI Industrial Automation, Apex, NC, USA</td>
			<td style="width:115px;"></td>
			<td style="width:160px;">Part of the OWT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHS Series Humidity&#160; Sensor Units</td>
			<td>&#160; TDK Corporation</td>
			<td></td>
			<td>Part of the OWT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature sensors</td>
			<td>(Murata NTSDXH103FPB30 thermistor) Murata Manufacturing Co., Ltd</td>
			<td></td>
			<td>Part of the OWT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LabVIEW 7.1.&#160;</td>
			<td>Laboratory Virtual Instrumentation Engineering Workbench, Version 7.1</td>
			<td></td>
			<td>Software Program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Self-Ligating brackets&#160;</td>
			<td>Empower Series, American Orthodontics.</td>
			<td></td>
			<td>Orthodontic Brackets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel archwires</td>
			<td>Ultimate Wireforms, Inc. in Bristol, CT</td>
			<td></td>
			<td>Archwires</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-Titanium Archwires</td>
			<td>Ultimate Wireforms, Inc. in Bristol, CT</td>
			<td></td>
			<td>Archwires</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Data acquisition device (DAQ)</td>
			<td>National Instruments (NI) USB 6210</td>
			<td></td>
			<td>Part of the OWT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ortho Form III (Archform template)</td>
			<td>3M Oral Care, St. Paul, MN, USA</td>
			<td></td>
			<td>Ovoid arch form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weingart Plier</td>
			<td>Hu-Friedy Mfg. Co., LLC Chicago, IL</td>
			<td></td>
			<td>Orthodontic Plier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light wire Plier</td>
			<td>Hu-Friedy Mfg. Co., LLC Chicago, IL</td>
			<td></td>
			<td>Orthodontic Plier</td>
		</tr>
	</tbody>
</table>

force system with vertical v-bends: a 3d in vitro assessment of elastic and rigid rectangular archwires

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing the complicated multi-bracket appliances to a simple two-bracket system for the purpose of force system evaluation will be the first step in this direction. However, much of the orthodontic biomechanics in this regard is confined to 2D experimental studies, computer modeling/analysis or theoretical extrapolation of existing models. The objective of this protocol is to design, construct and validate an in vitro 3D model capable of measuring the forces and moments generated by an archwire with a V-bend placed between two brackets. Additional objectives are to compare the force system generated by different types of archwires among themselves and to previous models. For this purpose, a 2 x 4 appliance representing a molar and an incisor has been simulated. An orthodontic wire tester (OWT) is constructed consisting of two multi-axis force transducers or load cells (nanosensors) to which the orthodontic brackets are attached. The load cells are capable of measuring the force system in all the three planes of space. Two types of archwires, stainless-steel and beta-titanium of three different sizes (0.016 x 0.022 inch, 0.017 x 0.025 inch and 0.019 x 0.025 inch), are tested. Each wire receives a single vertical V-bend systematically placed at a specific position with a predefined angle. Similar V-bends are replicated on different archwires at 11 different locations between the molar and incisor attachments. This is the first time an attempt has been made in vitro to simulate an orthodontic appliance utilizing V-bends on different archwires.

The total force and total moment experienced by each sensor at the center of the sensor plate are represented by their three orthogonal components: Fx, Fy, and Fz representing the forces along the x-axis, y-axis, and z-axis, respectively; and Mx, My, and Mz representing the moments around the same axes. The initial measurements at the sensors are converted mathematically to the force and moment values experienced by the bra...

Watch this Scientific Journal Video about Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires at JoVE.com

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

The method presented here is designed to construct and validate an in vitro 3D model capable of measuring the force system generated by different archwires with V-bends placed between two brackets. Additional objectives are to compare this force system with different types of archwires and to previous models.

Force System with Vertical V-Bends: A 3D In Vitro Assessment of Elastic and Rigid Rectangular Archwires

A proper understanding of the force system created by various orthodontic appliances can make treatment of patients efficient and predictable. Reducing ...

The apparatus described reduces a normal clinical set of 12 brackets to only two. Now this greatly simplifies our understanding of how an appliance works. It also provides a blueprint for testing and making modifications on similar appliances.This experiment answers some key questions related to the variation and force system created by V-bends. When placing those bands at different locations along the incisor molar arch span. The implications of this major apparatus is tend toward creating orthodontic appliances with a more predictable force system, which, at a clinical level would imply more predictable outcomes.To begin this procedure, insert a 0.021 by 0.025 inch SS ovoid maxillary archwire into the bracket slots. Then, place the testing apparatus in the glass chamber. Check for any unintended archwire activation.Any activation of the archwire will automatically create a force system, which will be displayed on the computer screen. If any archwire activation is observed, reposition the brackets, and check again. To fabricate a template archwire, place an archwire in the testing apparatus.Then, use a permanent marker to indicate the midline, a point immediately distal to the incisor bracket, and a point immediately mesial to the molar tube. Do the same for the contralateral side of the archwire. Next, transfer the archwire with the marked points to graph paper.Make a precise replica of the archwire on it, which can be used to determine the position of the V-bend for all archwires of the sample. Then, calculate the perimeter of the archwire segment L from I to M.Now, mark 11 points from I to M.Each point is a future V-bend position. Label each point from a0 to a10.Make sure that each bend position is separated from the other by an equal amount. Obtain a unique number for each bend position by calculating the a/L for each position. To place the V-bends, take a new archwire from the sample and place it on the template graph paper.Then, transfer one of the 11 bend positions bilaterally to the archwire. Use a rectangular archwire plier, or a light wire plier, to make symmetrical V-bends at both sides. Place the archwire on a flat platform and check the angle made by the two ends of the archwire with a protractor.Adjust the ends if necessary, so that an angle of 150 degrees is created. Subsequently, repeat the procedures for all archwires of the sample. To measure the force system, open the software program for data recording.Create a new folder for the data to be saved. Then, click Run to start the software. The program will display each of the three forces and three moment values at each sensor in real time.Wait for approximately 10 to 15 seconds for the fluctuations in data recording software to stop. Ensure that the graph lines for all the components of the force system show a flat line. Note that all six measurements at each sensor will show negligible values.Gently remove the testing apparatus from the platform. Use a Weingart plier to insert an archwire into the molar tubes. Next, open the door of the incisor bracket with a periodontal scaler.Lift the anterior portion of the archwire and insert it into the bracket slots. Make sure that the midline of the archwire coincides with the midline of the testing apparatus. After placing the archwire in the bracket slots, if the archwire has to be taken off for some adjustments, then the same archwire cannot be used and a new archwire will have to be inserted.Afterward, return the testing apparatus to the platform and close the door of the glass chamber. Set the temperature at thirty-seven degrees Celsius, and wait one minute for the temperature of the glass chamber to adjust. Click the Start Saving button on the software and allow the software to save and transfer data for at least 10 seconds.Click the Start Saving button again to end data transfer, then click STOP. Note that each measurement cycle generates 100 readings over the 10-second period for each component. Repeat the procedures for the 10 archwires of that specific bend position, and for all bend positions and types of archwires.To evaluate the error, open the software program for data recording. Create a new folder for the data to be saved. Then, click Run to start the software.The program will display each of the three forces, and three moment values at each sensor, in real time. Wait for approximately 10-15 seconds for the fluctuations in data recording software to stop. Ensure that the graph lines on the software for all the components of the force system show a flat line.Remove the testing apparatus from the platform. Using a light wire plier, bend one end of a straight length of a 0.021 x 0.025 in SS wire into a small hook. Insert the free end of the archwire into the molar tube from the distal side.Next, place the testing apparatus back on the platform. Attach a known weight to the hook and let it hang freely in the vertical plane by removing any type of interference. Close the door of the glass chamber.Click the Start Saving button on the software and allow the software to save and transfer data for at least 10 seconds. Click the Start Saving button again to end data transfer, then click STOP. Go to the document containing the saved data, export the data set to a custom designed data analysis spreadsheet, and repeat the error evaluation procedures for the incisor bracket.The vertical forces show symmetric and linear pattern for each of the six wire types. As the V-bend gets closer to either bracket, the vertical forces get higher. As the bend is moved farther beyond this point, the vertical force progressively increases.However, the directions of the individual vertical forces are reversed. Stainless steel archwires create a significantly greater force system than beta titanium archwires. Surprisingly, the relative force system created at the two brackets by the archwires, both in terms of size and type of archwire, is quite similar.In contrast, the moments show a nonlinear and asymmetric pattern. The flattening of Mxi, when V-bends are placed close to the molar tube, as well as the reversal of the moment direction in the molar tube from ax/L of 0.0 to 0.2 was similar for all archwires, and perhaps, represents a more fundamental nature of archwire bracket interaction and bracket orientation. The ratio of the moment of the two brackets shows some specific patterns observed across all archwires tested, and can be grouped into three distinct patterns.This approach can be utilized in numerous other clinical scenarios besides just V-bends. We have already begun testing some of those, and in the near future, we are looking at integrating this data directly with FEM simulations for similar setups. This approach will help in understanding and predicting clinical outcomes in patients.This will be a huge step in customizing treatment according to patient needs.

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Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...