Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Summary

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Abstract

Implantable microdevices are gaining significant attention for several biomedical applications^1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings^5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain^7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties^10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo¹⁵, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control^13,16,17.

To this end, a custom microtensile tester was designed to accommodate microscale samples^13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling¹⁷. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.

The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Protocol

1. Sample Preparation

1. Prepare PVAc-NC film of thickness in the range of 25-100 μm using a solution casting and compression technique^10-12.
2. Adhere film to a silicon wafer by heating on a hot plate for two min at 70 °C (above the glass transition temperature) to promote intimate contact between the film and the wafer. This step ensures that the prepared film remains flat and fixed to the Si wafer, which is necessary for planar micromachining processes.
3. Pattern the film into the test sample geometries by laser-micromachining (VLS 3.50, VersaLASER). Set the CO₂ direct-write laser micromachining parameters to 1.0% power (0.5 W), 4.0% speed (56 mm/s), and 1,000 pulses per inch^13,16.
4. Pattern samples that will be used to establish environmental conditions ("setup samples") into dogbone-shaped structures with lateral pad dimensions 1.5 x 1.5 mm², and lateral beam dimensions 300 x 3,000 μm², with a thickness matching that of the film throughout (Figure 2).
5. Pattern the samples for ex vivo experiments ("implant sample") into beams 300 μm x 6 mm, with a thickness matching that of the film.
6. Carefully release the samples from the wafer using a razor blade and tweezers.
7. For sample handling, prepare custom-machined acrylic holders designed to serve as part of the grip system in the microtensile tester. Laser-etched markings show the centerline of the holder and 1.5 mm from the end. Place a small amount of cyanoacrylate gel-based adhesive on the centerline of the acrylic holder and carefully adhere a 1.5 mm length of the implant sample to the holder and overlap the marked centerline (Figure 3). Each implant sample requires one acrylic holder. Be careful to ensure that the adhesive gel remains only along the 1.5 mm length of PVAc-NC being adhered to the acrylic holder. Otherwise, the adhesive gel can interfere with the mechanical behavior of the sample.
8. Remove moisture from all samples by placing them in a desiccator for at least 24 hr.
9. Measure the length, width, and thickness dimensions of the samples using an optical microscope.

2. Establish Environmental Conditions

1. Load a dry setup sample into the microtensile tester (see Figure 4), first clamping between the mobile grips, then between the fixed grips.
2. Mount an air brush with a water-filled reservoir into a fixed position, with the nozzle directed toward the microtensile sample. Connect the air brush to an air compressor via plastic tubing. With the air brush nozzle completely closed, turn on the air compressor.
3. Begin cyclic microtensile testing procedure, alternating between tensile strain (positive strain) and compressive strain (negative strain) applied to the sample, remaining within the linear elastic region of the stress-strain plot. For PVAc-NC, the applied strain is limited to less than 2%. In the custom microtensile tester used in these experiments, the strain rate was controlled while the required force to achieve that strain was measured. Alternatively, a different setup could involve controlling the applied force while measuring the resultant strain.
4. Gradually increase the flow from the air brush nozzle, and monitor the slope of the stress-strain plot as a function of the amount of flow from the air brush. The maximum flow that does not cause a significant (>10%) reduction in Young's modulus over a period of 60 sec is the level that will be used for the ex vivo experiments. At this point, the humidity conditions that will not wet a dry sample (and thus contribute to a reduction in Young's modulus), and will also minimize sample drying after being exposed to biological fluids in vivo have been established.
5. Measure the temperature near the sample. An ideal setup would include a thermocouple with digital readout, and be performed while the airbrush is operating. Set the intensity and distance of the radiant heat source such that the sample temperature is held to 37 °C, to match physiological conditions.

3. Compare Environmental Control to Non-Environmental Control

1. Immerse setup samples for at least 30 min in phosphate buffered saline. After this amount of time, the sample is completely saturated and has been reduced to its minimum Young's modulus at a given temperature.
2. Quickly load a sample into the microtensile tester and begin cyclic microtensile testing, with the air brush off, while the sample dries. This will determine how quickly the sample dries under non-controlled conditions.
3. Load a second PBS-saturated setup sample into the microtensile tester, and begin cyclic microtensile testing with the air brush on. This will determine how quickly the sample dries under controlled environmental conditions.

4. Probe Implantation and Explantation

1. Attach implant sample to a micromanipulator clamp and position orthogonal to the cortical tissue.
2. Prior to insertion, keep tissue sufficiently moist with saline to ensure homogeneity of tissue mechanics.
3. Lower the polymer sample into the cortex using the micromanipulator hand controls. Leave sample in cortical tissue until the target implant time, generally between 1 and 30 min. To prevent tissue from drying for time points over 5 min, lightly dab the tissue every 5 min using a saline-soaked cotton swab.
4. While the probe is implanted in the cortex, prepare the microtensile tester for loading the currently implanted sample by setting the drive rod to the zero-displacement position of 3.0 mm from the stationary sample clamp. Also, set the air brush nozzle to the flow setting and the radiant heat source to the proper intensity determine in Step 2.4.
5. At the end of the specified implant time, raise the probe out of the cortex using the micromanipulator hand controls. Immediately, and carefully, remove the sample from the micromanipulator clamp and load into the microtensile tester, as described in more detail in Step 5.2.

5. Microtensile Testing of Implant Samples

1. To save time after explantation, ensure that the microtensile tester is completely ready to accept the implant sample prior to implantation, as described in Step 4.4.
2. Immediately after explantation, load the sample between the two sets of microtensile tester clamps. Since the sample is mounted to an acrylic holder designed to serve as the top half of one clamp, place the implant sample assembly on the mobile grip, sample side down. It is important to ensure that the sample is mounted such that strain is applied only along the length of the probe to avoid applying torque to the sample during testing. As such, the sample must be mounted to the center of each clamp, and the clamps must be level with respect to each other.
3. Adjust the sample position such that the distance between the clamps is 3.0 mm, and the end of the probe is placed into the fixed clamp. This 3.0 mm length between clamps is the gauge length for the sample, and will be used in later calculations to determine the strain on the sample.
4. Immediately after securing the sample between both clamps, and within 2 min of explantation from the neural tissue, activate the motor in the tensile direction to elongate the sample at a constant rate (10 μm/sec used here), while simultaneously measuring and recording the elongation of the sample (using a displacement indicator, Mitutoyu 543-561) and associated force (using a load cell, Transducer Techniques MDB-2.5) required to strain the sample.
5. Halt the microtensile test upon mechanical failure of the sample, or when the end of the drive rod range is reached. Export the collected data, and perform data analysis.
6. Repeat the microtensile testing for each sample and/or each set of conditions (i.e. insertion time).

6. Data Analysis

1. Convert the raw elongation data to engineering strain applied to the implant sample by dividing the distance of elongation by the initial gauge length, as described in Equation 1, where ε is the applied strain, t is time, d is the displacement measured by the micrometer indicator, and L₀ is the initial gauge length of the sample:
   (1)
2. Convert the raw force data to the engineering stress on the sample by dividing the force (in Newtons), by the transverse cross-sectional area, as described in Equation 2:
   (2)
   where σ is the stress on the sample, F is the force measured by the load cell (in Newtons), w₀ is the initial width of the sample, and t₀ is the initial thickness of the sample.
3. Plot the stress (σ[t]) vs. strain (ε[t]) curve for each sample using a computer program, such as Microsoft Excel.
4. Isolate the linear elastic portion of the plot and use software-based curve fitting tools to find the best fit line to this portion. The slope of the best fit line corresponds to the Young's modulus of the sample. The isolated portion of the plot should include at least 10 stress-strain points, and should be taken from the portion of the plot where the slope is greatest.
5. For cyclic tests, the Young's modulus will need to be determined for each cycle. This may be automated or performed manually.
6. For the cyclic tests, plot the Young's modulus of each cycle versus time. This indicates how the measured modulus changes with time, which is indicative of how quickly a setup sample is wetting or drying.
7. For implant samples, each sample and time of implant corresponds to a single cycle of the cyclic tests. Measure the Young's modulus using the procedure described above for each implant sample.
8. Plot the Young's modulus versus implant time. At this point, comparisons can be made to benchtop investigations, etc.

Results

The mechanical properties of nearly all polymeric materials, including our PVAc-NC, are dependent upon exposure to environmental conditions. Most notably, these include the exposure to heat and moisture. When a material is plasticized due to the uptake of moisture, or undergoes a thermal transition, it displays a reduction in Young's modulus. In preparing the moisture- and temperature-controlled environment for ex vivo sample mechanical characterization, it is important to ensure that there is minimal change in...

Discussion

The advancement of implantable biomedical microelectromechanical systems (bioMEMS) for interacting with biological systems is motivating the development of new materials with highly-tailored properties. Some of these materials are designed to exhibit a change in material properties in response to a stimulus found in the physiological environment. One recently-developed class of materials responds to the presence of hydrogen bond-forming liquids (e.g. water) and elevated temperatures to reduce the Young's modulus...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicon wafer	University Wafer		Mechanical grade
Extruded acrylic sheet	Professional Plastics	SACR 062EF	Thickness 0.062"
Razor blade	McMaster-Carr	3962A3
Tweezers	McMaster-Carr	8384A47	#5 tip
Super Glue Gel	Loctite	130380
Air Brush	Snap-on Industrial	BF175TA
Air Compressor	Paasche	B002YKN8YO	D500
Thermocouple	Omega	HH12A
Hot plate	Cimarec	SP131325Q
CO2 direct-write laser	VersaLaser	3.5
Dessicator	Fisher Scientific	08-595
Lamp			custom-built
Microtensile tester			custom-built