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

The overall goal of this procedure is to quantify the change in stiffness on a polyvinyl, acetate based nano composite structure as a function of time implanted in tissue. This is accomplished by first patterning the polymer nano composite samples and adhering them to acrylic holders for insertion into tissue and micro mechanical testing. The second step is to prepare a micro tensile tester environment to mimic the physiological environment, ex vivo using a humidity source and a radiant heat source.

Next, the implant sample is inserted into tissue and removed after a specified duration. The final step is to load the sample into the environmentally controlled micro tensile tester and perform the mechanical testing to determine the young's modulus of the material after the specified implant duration. Ultimately, this environmentally controlled micro tensile testing is used to show the changes in mechanical stiffness as measured by Young's modulus as a function of time exposed to the physiological environment.

The main advantage of this technique over existing methods of measuring variable mechanical properties like dynamic mechanical analysis, is that it can be applied to microscale samples and the humidity and temperature can be controlled. This method can help answer key questions in the neural interfacing field, such as how the inflammatory response to an implant is affected by the stiffness of the implant material. First, obtain polyvinyl acetate based nano composite film with a thickness of 25 to 100 micrometers produced with a solution casting and compression technique.

Next, adhere the film to a silicone wafer by heating on a hot plate at 70 degrees centigrade for two minutes. This promotes intimate contact between the film and the wafer and ensures the film remains flat for micro machining. Now use laser microm machining to pattern the film into the desired test sample geometries.

The direct right laser micro machining parameters are set to a power of 0.5 watts, a speed of 56 millimeters per second, and 1000 pulses per inch machine. The samples that will be used to establish environmental conditions called setup samples into dog bone shaped structures with lateral pad dimensions of 1.5 millimeters by 1.5 millimeters and lateral beam dimensions of 300 micrometers by 3000 micrometers. The thickness matches that of the film throughout machine the samples for ex vivo experiments referred to as implant samples into beams, 300 micrometers by six millimeters with a thickness matching that of the film.

After removing the wafer from the micro machining setup, use a razor blade and tweezers to carefully release the samples from the wafer to handle the samples. Prepare acrylic holders designed to serve as part of the grip system in the micro tensile tester. In this experiment, each holder is 11 millimeters by seven millimeters with a thickness of 2.2 millimeters and has two holes to align with bolts in the micro tensile tester.

Laser etched markings showed the center line of the holder at 1.5 millimeters from the end. Each implant sample requires one acrylic holder, place a small amount of Sano acrylic gel-based adhesive on the center line of the acrylic holder, and carefully adhere a 1.5 millimeter length of the implant sample to the holder overlapping the marked center line. Be careful to ensure that the adhesive gel remains only along the 1.5 millimeter length of the polyvinyl acetate based nano composite being adhered to the acrylic holder.

Begin by loading a dry setup sample into the micro tensile tester. First clamping between the mobile grips, then between the fixed grips mount an airbrush with a water-filled reservoir into a fixed position with the nozzle directed toward the micro tensile sample. Connect the airbrush to an air compressor via plastic tubing with the airbrush nozzle completely closed.

Turn on the air compressor. Begin the cyclic micro tensile testing procedure, alternating between tensile strain and compressive strain being applied to the sample. The test should remain in the linear elastic region of the stress strain plot.

In this case, the applied strain is limited to less than 2%In these experiments, the strain rate was controlled while the required force to achieve that strain was measured. To determine the desired humidity conditions gradually increase the flow from the airbrush nozzle and monitor the slope of the stress train plot as a function of the amount of flow from the airbrush, the maximum flow that does not cause a significant reduction in young's modulus over a period of 60 seconds is the level that will be used for the ex vivo experiments. Finally, measure the temperature near the sample.

An ideal setup would include a thermocouple with a digital readout, and measurements would be performed while the airbrush is operating. Set the intensity and distance of a radiant heat source such that the sample temperature is held at 37 degrees Celsius to match physiological conditions. The control comparison begins by immersing the setup samples for at least 30 minutes in phosphate buffered saline or PBS to allow it to soften to its minimum Young's modulus.

Quickly load a sample into the micro tensile tester and begin cyclic micro tensile testing with the airbrush off while the sample dries under ambient conditions, the Young's ModuLite found from this data will indicate how quickly the sample dries under non-controlled conditions. Next, load a second PBS saturated setup. Sample into the micro tester and begin cyclic micro tensile testing with the airbrush on here, the calculated Young's mod, I will indicate how quickly the sample dries under controlled humidity conditions.

Secure a sample of cortical tissue. In this demonstration, the explanted tissue is kept hydrated in a bath of artificial cerebral spinal fluid maintained at 37 degrees Celsius before and throughout the experiment. Next, attach an implant sample on its holder to a micro manipulator clamp.

Position the probe so that it is orthogonal to the cortical tissue. Lower the polymer sample into the cortex using the microm manipulator. Hand controls.

Leave the sample in the cortical tissue until the target implant time has elapsed between one and 30 minutes. Take precautions against the tissue drying during this time while the probe is implanted in the cortex. Prepare the micro tensile tester by setting the dry rod to the zero displacement position of three millimeters from the stationary clamp.

Also set the airbrush nozzle flow setting and the radiant heat source power setting to the values determined previously. At the end of the specified implant time, raise the probe out of the cortex using the microm manipulator hand controls immediately and carefully remove the sample from the microm manipulator clamp and take it to the micro tensile tester to begin testing within two minutes immediately after explanation, load the sample between the two sets of micro tensile tester clamps. Since the sample holder is designed to serve as the top half of one clamp, place the implant sample assembly on the mobile grip sample side down.

The sample must be mounted to the center of each clamp, and the clamps must be level with respect to one another. This ensures that strain is only applied along the length of the probe. Now adjust the sample position such that the distance between the clamps is three millimeters and the end of the probe is placed into the fixed clamp.

The three millimeter length between the clamps is the gauge length of the sample to be used in later calculations. Immediately after securing the sample between both clamps and within two minutes of explanation from the neural tissue, activate the motor in the tensile direction to elongate the sample at a constant rate. The rate here is 10 micrometers per second.

Simultaneously measure and record elongation of the sample and the associated force required to strain the sample. Halt the micro tensile test upon mechanical failure of the sample or when the dry rod range is reached. Export the collected data for analysis.

Repeat the micro tensile testing for each sample and or implantation condition. Plot the stress versus strain curve for each sample using software. Next, isolate the linear elastic portion of the plot.

The isolated portion of the plot should include at least 10 points and should be taken from the section of the plot where the slope is greatest. Now 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.

For the setup samples that have been tested in cyclic mode, determine the Young's modulus for each cycle. Once this is done, plot the Young's modulus of each cycle versus time. This plot chose the Young's modulus as a function of time, as measured during cyclical tensile tests to determine the correct airbrush settings for controlling the environment.

The shaded region is the time during which the airbrush was turned on at the airbrush settings used. The Young's modulus does not change significantly over time. This suggests that the amount of water absorbed by the setup sample from the airbrush is not enough to contribute to a reduction in stiffness.

Here is the Young's modulus versus thyme for water saturated samples in both moisture controlled and non-controlled tensile testing environments. The recovery of the initial Young's modulus is much slower in the controlled environment. This demonstrates the increase in the time required for the sample to dry In this environment, this extra time can be used to perform mechanical tests on samples that have been implanted.

These are representative plots showing the stress train curves for a dry sample and a wet sample that had been implanted in a rat cortex for 30 minutes. The Young's modulus, which corresponds to the slope of the stress train plot in the linear elastic region, is clearly much greater for the dry sample. Both samples were strained to break in.

This final plot is the Young's modulus versus implant duration for the samples placed in the cortex. After about five minutes of implantation, the sample displays little change in the Young's modulus. This suggests that the sample reaches saturation and minimum stiffness within this period of time.

While attempting this procedure, it's important to remember to plan ahead to ensure that physiological conditions in both the tissue and ex vivo testing environment can be maintained. This method can be used to assess the mechanical behavior of other materials with environment dependent properties, including biodegradable materials and can be performed to assess the degradation rate or mechanical stability of polymeric materials in vivo.