The authors want to thank Dr. Taylor Ware for allowing us to use his environmental DMA.
This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program [W81XWH-15-1-0607]. Opinions, interpretations, conclusions, and recommendations are those of the authors and not necessarily endorsed by the Department of Defense.

1. Preparation of polymer samples for testing
<ol>
	<li>Synthesize the softening thiol-ene polymer according to previous protocols inside a fume hood.1,2,4,18 Briefly, mix quantitative amounts of thiol to alkene monomers with a total of 0.1 wt% photo initiator.
	<ol>
		<li>Prepare a 20 mL glass vial for polymer mixing. Cover the vial in aluminum foil to prevent incident light from contacting ...

<ol>
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L., Voit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart+Polymers+for+Neural+Interfaces.">Smart Polymers for Neural Interfaces.</a> Polymer Reviews. 53 (1), 108-129 (2013).</li><li>Ware, T., 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=Fabrication+of+Responsive,+Softening+Neural+Interfaces.">Fabrication of Responsive, Softening Neural Interfaces.</a> Advanced Functional Materials. 22 (16), 3470-3479 (2012).</li><li>Stiller, A. M., 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=Chronic+Intracortical+Recording+and+Electrochemical+Stability+of+Thiol-ene/Acrylate+Shape+Memory+Polymer+Electrode+Arrays.">Chronic Intracortical Recording and Electrochemical Stability of Thiol-ene/Acrylate Shape Memory Polymer Electrode Arrays.</a> Micromachines. 9 (10), 500 (2018).</li><li>Biran, R., Martin, D. C., Tresco, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cell+loss+accompanies+the+brain+tissue+response+to+chronically+implanted+silicon+microelectrode+arrays.">Neuronal cell loss accompanies the brain tissue response to chronically implanted silicon microelectrode arrays.</a> Experimental Neurology. 195 (1), 115-126 (2005).</li><li>Polikov, V. S., Tresco, P. A., Reichert, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+brain+tissue+to+chronically+implanted+neural+electrodes.">Response of brain tissue to chronically implanted neural electrodes.</a> Journal of Neuroscience Methods. 148 (1), 1-18 (2005).</li><li>Lacour, S. P., Courtine, G., Guck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+and+technologies+for+soft+implantable+neuroprostheses.">Materials and technologies for soft implantable neuroprostheses.</a> Nature Reviews Materials. 1 (10), 16063 (2016).</li><li>Stiller, A., 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=A+Meta-Analysis+of+Intracortical+Device+Stiffness+and+Its+Correlation+with+Histological+Outcomes.">A Meta-Analysis of Intracortical Device Stiffness and Its Correlation with Histological Outcomes.</a> Micromachines. 9 (9), 443 (2018).</li><li>Lecomte, A., Descamps, E., Bergaud, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+mechanical+considerations+for+chronically-implanted+neural+probes.">A review on mechanical considerations for chronically-implanted neural probes.</a> Journal of Neural Engineering. 15 (3), 031001 (2018).</li><li>Nguyen, J. K., 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=Mechanically-compliant+intracortical+implants+reduce+the+neuroinflammatory+response.">Mechanically-compliant intracortical implants reduce the neuroinflammatory response.</a> Journal of Neural Engineering. 11 (5), 056014 (2014).</li><li>Ecker, M., 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=From+Softening+Polymers+to+Multi-Material+Based+Bioelectronic+Devices.">From Softening Polymers to Multi-Material Based Bioelectronic Devices.</a> Multifunctional Materials. , (2018).</li><li>Hess, A. E., Potter, K. A., Tyler, D. J., Zorman, C. A., Capadona, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmentally-controlled+Microtensile+Testing+of+Mechanically-adaptive+Polymer+Nanocomposites+for+ex+vivo+Characterization.">Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization.</a> Journal of Visualized Experiments. (78), e50078 (2013).</li><li>Black, B. J., 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=In+vitro+compatibility+testing+of+thiol-ene/acrylate-based+shape+memory+polymers+for+use+in+implantable+neural+interfaces.">In vitro compatibility testing of thiol-ene/acrylate-based shape memory polymers for use in implantable neural interfaces.</a> Journal of Biomedical Materials Research Part A. 106 (11), 2891-2898 (2018).</li><li>Hassler, C., Boretius, T., Stieglitz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymers+for+neural+implants.">Polymers for neural implants.</a> Journal of Polymer Science Part B: Polymer Physics. 49 (1), 18-33 (2011).</li></ol>

The use of environmental DMA allows the study of the behavior of various polymers used as substrates for neural implants19 or other biomedical devices in solution and to mimic in vivo conditions. This includes, but is not limited to, polyimide, parylene-C, PDMS, and SU-8. Hydrogels and extracellular matrix (ECM) materials can also be investigated using this method. The differences of overall softening of the polymer as well as its softening kinetics can be easily compared between differen...

A new generation of materials used as substrates for neural implants comprises softening shape memory polymers1,2,3,4,5,6,7,8,9. These materials are stiff enough during implantation to overcome critical buckling forces, but they become up to three orders of magnitude softer after implantation in a body environment. It is predicted that these materials show a better device-tissue interaction due to the reduced mismatch in modulus as compared to traditional materials used in neural implants, such as tungsten or silicon. Traditional, stiff devices show inflammatory response after implantation, followed by tissue encapsulation and astroglial scarring which often results in device failure10,11. It is a common assumption that less stiff devices minimize the foreign body response12,13,14. The stiffness of a device is dictated by its cross-sectional area and modulus. Therefore, it is important to reduce both factors to improve the device compliance and, ultimately, the device tissue interaction.
The work on softening polymers was inspired by the work of Nguyen et al.15, who demonstrated that mechanically-compliant intracortical implants reduce the neuroinflammatory response. They have previously used mechanically-adaptive poly(vinyl acetate)/tunicate cellulose nanocrystal (tCNC) nanocomposites (NC), which become compliant after implantation.
The Voit lab, on the other hand, uses the highly tunable system of thiol-ene and thiol-ene/acrylate polymers. These materials are advantageous in that the degree of softening after exposure to in vivo conditions can easily be tuned by the polymer design. By choosing the right polymer composition and crosslink density, the glass transition temperature and Young&#39;s modulus of the polymer can be modified2,4,5,6,8. The underlying effect of the softening is the plasticization of the polymer in an aqueous environment. By having a polymer with a glass transition temperature (Tg) above body temperature when dry (the state during implantation), but below body temperature after being immersed in water or PBS, the resulting stiffness/modulus of the polymer can shift from glassy (stiff) when dry to rubbery (soft) when implanted16.
However, exact and reliable measurements of the softening due to plasticization and the shift of Tg from the dry to wet states have not been able to be measured in the past. Traditional dynamic mechanical analysis is performed in air or inert gases and does not allow for measuring of the thermomechanical properties of polymers inside a solution. In previous studies, the polymers have been immersed in PBS for various amounts of time. Swollen samples were then used to perform dynamic mechanical analysis (DMA)6,7,8. However, since the procedure involves a temperature ramp, samples start to dry during the measurement and do not yield representative data. This is especially true if the sample size becomes smaller. In order to predict the softening of neural probes, it would be necessary to test 5 to 50 &#181;m-thin polymer films, which is not possible with traditional DMA due to the abovementioned drying of the samples during the measurement.
Hess et al.17 have designed a custom-built microtensile testing machine to assess the mechanical properties of mechanically adaptive materials using an environmentally controlled method. They have previously used an airbrush system to spray water on samples during the measurement to prevent them from drying out.
The use of environmental DMA (Figure 1), however, allows for measurement of polymer films in solutions, such as water and PBS, at various temperatures. This allows not only measurement of the polymer&#39;s thermomechanical properties in the soaked/softened state but also measurement of its softening kinetics. Even tensile tests and swelling measurements are possible inside the immersion bath of this machine. This allows for exact studies of the plasticization-induced softening of polymer substrates to predict in vivo behaviors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,3,5-Triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO)</td>
			<td>Sigma-Aldrich</td>
			<td>114235-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2-Dimethoxy-2-phenylacetophenone (DMPA)</td>
			<td>Sigma-Aldrich</td>
			<td>196118-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 laser Gravograph LS100</td>
			<td>Gravotech, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Large Glass Microscope Slides, 75 x 50mm</td>
			<td>Ted Pella</td>
			<td>26005</td>
			<td></td>
		</tr>
		<tr>
			<td>Environmental DMA: RSA-G2 Solids Analyzer</td>
			<td>TA Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ESD Safe Plastic Tweezer, Tips; Flat, Duckbill, 11.5 cm</td>
			<td>Cole Palmer</td>
			<td>EW-07387-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Laurell WS-650-8B spin coater</td>
			<td>Laurell Technologies Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Air gas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 1X Solution, Fisher BioReagents</td>
			<td>Fisher Scientific</td>
			<td>BP243820</td>
			<td></td>
		</tr>
		<tr>
			<td>SHEL LAB vacuum oven</td>
			<td>VWR International</td>
			<td>89409-484</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td></td>
			<td>Mechanical grade</td>
		</tr>
		<tr>
			<td>The RSA-G2 Immersion System</td>
			<td>TA Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trimethylolpropane tris(3-mercaptopropionate) (TMTMP)</td>
			<td>Sigma-Aldrich</td>
			<td>381489-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP CL-1000 crosslinking chamber with 365 nm bulbs</td>
			<td>VWR International</td>
			<td>21474-598</td>
			<td></td>
		</tr>
	</tbody>
</table>

environmental dynamic mechanical analysis to predict the softening behavior of neural implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

The use of environmental DMA allows the analysis of softening kinetics and overall softening capabilities of polymers. By using the temperature-time measuring mode of the protocol, the softening profiles of different polymer formulations can be compared to each other (Figure 6). This method can also be used to quantify softening and swelling rates of polymers. It can be seen in Figure 4 that different polymer formulations may und...

Watch this Scientific Journal Video about Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants at JoVE.com

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

This method helps in monitoring how the material properties of implantable devices may change upon insertion into the body, particularly the softening of the materials. The main advantage of this technique is that it offers a simple in vitro method that mimics in vivo conditions and therefore minimizes the need for animal testing. This method is especially interesting for testing the thermomechanical properties of the material that will be used in implantable devices in general and is not limited to bioelectronics.The loading of the sample and the measurement of the sample under the wet conditions are a little bit tricky and may cause unreliable test results if not performed properly. Begin by mixing quantitative amounts of thiol to alkene monomers with a total of 1 weight percent photoinitiator. Cover a 20-milliliter glass vial in aluminum foil to prevent incident light from contacting the monomer solution, and use a disposal plastic pipette to add 50 mole percent TATATO, 45 molar percent TMTMP, and five molar percent TMICN to the vial.Then add 1 weight percent of the photoinitiator DMPA to the vial, and use planetary speed mixing to mix the contents of the vial without exposing the solution to light. When the contents have been thoroughly mixed, spin coat the resulting thiol-ene prepolymer mixture into glass microscope slides in five 50-micrometer-thick films, and immediately transfer the polymer films on the carrier substrate in a crosslinking chamber. Then photo-polymerize the films for 60 minutes under 365-nanometer ultraviolet bulbs, followed by post-curing in a vacuum oven for 24 hours at 120 degrees Celsius to further complete the conversion.When the polymers have fully cured, use a carbon dioxide laser to cut the films into 4.5-millimeter-wide by 50-millimeter-long rectangles for dynamic mechanical testing. To set up the dynamic mechanical analyzer, equip the machine with the immersion fixture in tension mode. Connect the liquid nitrogen to the machine, and enable liquid nitrogen in air as a gas source for the furnace.Write the method for the dry measurement with the machine software, including the conditioning, oscillation temperature ramp, and conditioning end of the test steps. Then write the method for the immersion testing with the machine software, including the conditioning, oscillation time, oscillation temperature ramp, and conditioning end of test steps. When the analyzer is ready, use calipers with a 001-millimeter precision to measure the actual thickness of the polymer specimen for dry in air testing, and enter the sample name, description, and sample geometry into the software.Set the loading gap to 15 millimeters, and load the sample. Make sure to center and align the specimen before tightening the clamps. Then close the furnace, and start the dry measurement.When the measurement is over, open the furnace, and remove the polymer sample from the machine. To measure the actual thickness of the polymer specimen for immersion testing, first measure the sample with calipers with a 001-millimeter precision, and enter the sample name, description, and sample geometry into the software. Prepare the setup with the immersion beaker fixed with a clamp at the upper grip, and set the loading gap to 15 millimeters.Load the sample, making sure to center and align the specimen, and tighten the clamps. Place the immersion bath on the bottom fixture, and secure the bath tightly. Then fill the bath with room-temperature PBS.Place the lid on top of the bath, close the furnace, and immediately start the immersion measurement, confirming that the drain is closed. It's important to start the measurement as soon as possible after the beaker is filled with the PBS to ensure that the full range of the softening is captured. When the measurement is over, open the drain to remove the PBS from the immersion bath, and open the furnace.Then remove the lid from the beaker, unscrew and lift the immersion beaker, and remove the polymer sample from the machine. Using the temperature-time measuring mode of the protocol allows the softening profiles of different polymer formulations to be compared. Combining dry dynamic mechanical analysis measurements and immersion measurements in PBS allows the assessment of water-induced plasticization of different polymer formulations, as illustrated by the depression of the glass transition temperature and overall downshift of the modulus curves.The softening of the polymers for in vivo applications works most effectively when the dry polymer has a glass transition temperature above body temperature but below that in the wet state. Thus, the modulus of the polymer drops from the glassy to rubbery modulus upon immersion under physiological conditions. When the glass transition temperature of both the dry and wet states of the polymer are well above body temperature, the polymer will not soften under physiological conditions.This method allows you to replace the PBS with other relevant solutions to mimic the behavior of biomaterials in other environments.

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JoVE Journal

Bioengineering

7.5K vistas.  University of Texas at Dallas. Este método ayuda a controlar cómo las propiedades materiales de los dispositivos implantables pueden cambiar al insertarse en el cuerpo, particularmente el ablandamiento de los materiales. La principal ventaja de esta técnica es que ofrece un método simple in vitro que imita las condiciones in vivo y por lo tanto minimiza la necesidad de pruebas con animales. Este método es especialmente interesante para probar las propiedades termomecánicas del material que se utilizará en dispositiv...