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 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.
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'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 µm-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'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.
1. Preparation of polymer samples for testing
2. Machine setup
3. Sample loading and unloading for dry measurements
4. Sample loading and unloading for immersion testing
5. Measurements
6. Data interpretation
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 undergo different degrees of softening while being immersed in the 37 °C PBS. The non-softening version remains in the GPa range, whereas the semi-softening polymer softens from 1700 MPa to 370 MPa, and the fully softening polymer to 40 MPa. The softening of all three polymer formulations takes place within 10 to 15 min.
The use of the combination of dry DMA measurements and measurements in PBS allows the assessment of water-induced plasticization of different polymer formulations, which is shown by depression of the Tg and overall downshift of the modulus curves (Figure 7). The softening of the polymers is working most effectively when the dry polymer has a Tg 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 (Figure 7A). When the Tg of both the dry and wet states of the polymer are well above body temperature, the polymer will not soften under physiological conditions (Figure 7B).
Figure 1: Environmental DMA with immersion system. (A) A more detailed view of the fixture for dry (B) and wet (C) measuring conditions. (B) and (C) are previously published by Ecker et al.2. Please click here to view a larger version of this figure.
Figure 2: Spin curves for fully softening thiol-ene polymer. Spin curves for fully softening thiol-ene polymer showing the relationship between spin speed and time and the resulting film thickness. Please click here to view a larger version of this figure.
Figure 3: Fabrication of DMA test stripes on microscopic glass slides. Fabrication of DMA test stripes on microscopic glass slides (A) or silicon wafers (B) using photolithography. Please click here to view a larger version of this figure.
Figure 4: Sample loading for measurement with immersion bath. A () DMA equipped with immersion fixture, (B) immersion beaker temporarily fixed with clamps around upper grip, (C) loading of polymer sample at a clamp distance of 15 mm, (D) lowering of immersion beaker to lower fixture and fixation with screws, (E) filling the immersion beaker with PBS, (F) closing the lid, (G) closing the furnace, and (H) ensuring that drain is closed. Please click here to view a larger version of this figure.
Figure 5: Alignment of sample. (A) The sample must be straight and centered between the top and bottom clamps. Samples should not be diagonal (B), too high or too low (C), or too much toward the edges (D). Sample should also not be buckled (E) but should be straight (F) to ensure reliable measurements. Please click here to view a larger version of this figure.
Figure 6: Softening kinetics of three different thiol-ene polymers. Softening kinetics of three different thiol-ene polymers as measured with the oscillation-time protocol inside PBS at 37 °C for 1 h. Please click here to view a larger version of this figure.
Figure 7: Displays DMA measurements of two different SMP formulations. Displays DMA measurements of two different SMP formulations before (orange) and after (blue) soaking in PBS, respectively. (A) A fully-softening (FS) version and (B) slightly-softening version (SS) of SMP. This figure has been modified from Ecker et al.2. Please click here to view a larger version of this figure.
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 different solutions, including water, heavy water, and PBS. It is also possible to test the influence of different immersion temperatures or differences resulting from varying polymer thicknesses and compositions.
This method also allows studying of the influence of various treatments on softening behaviors of polymers and hydrogels. Treatments include application of various sterilization methods, accelerated aging in various media, and surface modification. This in vitro method will help researchers learn about the behavior and durability of these materials, obtain reliable in vitro measurements, and avoid unnecessary animal experiments. However, measuring in PBS is just one approach to mimic the biological environments. In vivo conditions may vary in many aspects, such as ion concentration and the availability of antibodies, proteins, and other species inside biological media/tissues. Depending on the targeted area, experimenters may also consider using different media for environmental measurements, such as tris-buffered saline (TBS), TBS-T (TBS with polysorbate 20), bovine serum albumin (BSA), cerebrospinal fluid (CSF), and other body fluids.
In addition, it is possible to characterize the mechanical properties of probes after explantation from an animal after an in vivo study is completed. This will allow the investigation of probe behavior after softening in a body environment and comparison to in vitro data.
It should be noted that there is an offset between the temperature set for the solution bath and the actual temperature. This is due to the fact that two different temperature controllers are being used: one for temperature control (outside the immersion bath) and another for measuring the temperature (inside the immersion bath). We found that when the outside temperature is set to 39.5 °C, the temperature inside the bath stabilized at 37 °C.
The temperature range for measurements inside solutions are naturally limited by their crystallization and boiling temperatures. It is recommended to remain at least 10 K above and below these temperatures, respectively.
It is debated whether the starting temperature of the immersion solution used for soaking/softening measurements should be room temperature or pre-warmed to body temperature to best mimic the conditions during probe implantation. The use of RT PBS takes into account the fact that the probe is kept at RT before implantation and that it is usually kept in close proximity to the implantation side while it is aligned to the right position. At this stage, the probe may already start to soften due to the moist milieu. Starting with 37 °C PBS will better mimic a shotgun approach for insertion.
The described results were measured on polymer films in tension mode; however, the environmental DMA is also capable of measurements in compression and in shear when using the respective fixture. Therefore, this also allows for the measurement of other sample geometries. It should be noted that the available space inside the immersion beaker is limited and thus the samples used for measurements inside this beaker are restricted by their sizes.
Another limitation of this method is the load cell, which is used to detect the forces generated by the samples during the measurement (in dry and wet conditions). The load cell can only measure forces up to 35 N, which therefore limits the sample size/geometry.
The authors have nothing to disclose.
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.
1,3,5-Triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) | Sigma-Aldrich | 114235-100G | |
2,2-Dimethoxy-2-phenylacetophenone (DMPA) | Sigma-Aldrich | 196118-50G | |
CO2 laser Gravograph LS100 | Gravotech, Inc. | ||
Corning Large Glass Microscope Slides, 75 x 50mm | Ted Pella | 26005 | |
Environmental DMA: RSA-G2 Solids Analyzer | TA Instruments | ||
ESD Safe Plastic Tweezer, Tips; Flat, Duckbill, 11.5 cm | Cole Palmer | EW-07387-17 | |
Laurell WS-650-8B spin coater | Laurell Technologies Corporation | ||
liquid nitrogen | Air gas | ||
PBS, 1X Solution, Fisher BioReagents | Fisher Scientific | BP243820 | |
SHEL LAB vacuum oven | VWR International | 89409-484 | |
Silicon wafer | University Wafer | Mechanical grade | |
The RSA-G2 Immersion System | TA Instruments | ||
Trimethylolpropane tris(3-mercaptopropionate) (TMTMP) | Sigma-Aldrich | 381489-100ML | |
UVP CL-1000 crosslinking chamber with 365 nm bulbs | VWR International | 21474-598 |