Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

To examine engineered tissue constructs traditional mechanical testing, which often results in the destruction of the sample is not acceptable. This method uses microscopic magnetic resonance elastography or micro MRE as a non-invasive technique for measuring the mechanical properties of small soft tissues. First cells are seated onto a biomaterial scaffold to generate tissue.

The tissue is suspended in aros gel, and the tip of an actuator is placed into the gel. Then to characterize the actuator motion is transferred to the sample and detected using a laser. Doppler viter.

The sample and actuator are transferred to a magnet and magnetic resonance. Elastography images are acquired. Analysis of the resulting images shows the change in sheer stiffness for both osteogenic and adipogenic constructs.

We first had the idea for this technique and we observed magnetic residency phtography for the use of disease diagnosis and realized it could be extended to tissue engineering. The main advantage of this technique over other existing techniques like mechanical testing, is that it applies the non-invasive technology of MRI to major mechanical properties of tissues. The applications of this technique extend to tissue engineering because the knowledge of mechanical properties will ensure it is appropriate for its intended uses in bone and cartilage engineering.

Although these methods provide valuable insights into tissue engineering, it can also be used to diagnose diseases in different organs, such as the liver fibrosis, traumatic brain injury, or brain cancer, for instance. Visual demonstration of this method is critical because it includes the steps which are difficult to learn, and prior awareness of tissue engineering and magnitude rhythm imaging.Gars. The tissue construct preparation process consists of three main stages, expansion of cell population, seeding of cells onto a biomaterial scaffold and differentiation through the use of chemical signaling molecules.

After culture and expansion of the cell line seed the human mesenchymal stem cells or HMCs onto a gelatin sponge at a density of one times 10 to the six cells per milliliter for bone formation. Approximately three days later, the cells should appear confluent on the scaffold to induce differentiation. Remove the medium and replace it with adipose induction medium.

Then incubate the cells at 37 degrees Celsius with 5%carbon dioxide after three days. Replace the medium with maintenance medium, which consists of expansion medium containing 10 micrograms per milliliter of human recombinant insulin. After incubating for 24 hours, replace the maintenance medium with induction medium.

Repeat this cycle three times. Replace the maintenance medium every two days thereafter for four weeks to induce osteogenesis. Replace with fresh osteogenic medium every two days for the duration of the study.

Here the study is four weeks long and MRE is performed each week. Magnetic resonance elastography relies on the propagation of mechanical shear waves to assess local values of mechanical properties. Therefore, these mechanical vibrations need to be generated and characterized within the tissue of interest using a piso electric actuator To prepare the sample, transfer the tissue culture to a 10 millimeter diameter test tube containing a solid base and a layer of 0.5%AGROS gel.

Then add warm 0.5 acro gel. To enclose it. Allow the agros gel to set for five minutes.

At room temperature, insert the tip of the piso electric bending motor into the surface of the gel. Next, attach the tube containing the sample and the actuator to a rigid support. Orient the beam of the laser doppler barometer toward the tip of the mechanical actuator.

Adjust the positioning of the system to optimize the reflected signal strength, which is displayed on the barometer. To maximize reflection, use reflective tape if necessary to set up the actuator to generate harmless sheer waves with significant amplitudes of about 250 microns. Set the function generator to sweep the desired frequency range using an operating voltage of 20 volts peak tope with a white noise signal.

For this experiment, the desired frequency range is 20 to 2000 hertz. To view the characterized spectrum on the Polytech Rsof program, select velocity and FFT display. Begin capturing signal and identify the resonance frequency of the system based on peaks of the spectrum.

Next, to measure the deflection of the actuator, set the actuator to deliver a continuous sinusoid at the characterized resonance frequency. Using an operating voltage of 200 volts peak tope and denote the generated displacement being delivered by the actuator to the surface of the gel set fiber soft to display the FFT with displacement as the Y axis. Once the actuator has been characterized, place the test tube containing the sample and actuator into a slot in a 10 millimeter RF coil, place the sample and actuator in the center of the MRI scanner.

Acquire a scout image for identification of the construct location. Once the tissue construct has been located, set the parameters for the acquisition. A typical in vitro sagittal scan will have a repetition time of 1000 milliseconds.

Echo time of 20 to 40 milliseconds slice thickness of 0.5 to one millimeter and field of view of 12 by 10 millimeter squared, with a matrix size of 1 28 by 1 28 pixels. For the elastography parameters, set the actuator frequency to the value determined by the laser doppler viter characterization. For this sample, one bi pair is needed with a gradient amplitude of 50 gauss per centimeter and an MRE delay set to zero.

Change the function generator to burst mode and adjust the parameters of the function generator to match those in the elastography acquisition parameters, including the frequency and number of cycles. Also set the function generator to be externally triggered. To obtain a sagittal image, set the motion sensitization to be in the positive slice direction and start the scan following acquisition.

Check the image to evaluate the signal quality in the tissue construct. If the image looks too dark, adjust the MR parameters and acquire another scan. Next, change the sensitization to the negative slice direction.

Transfer the files from the MRI scanner to another computer equipped with MATLAB and execute the MATLAB program that will perform a complex division for generation of an image depicting shear waves propagation. Assess the image for the presence of shear waves and possible artifacts such as phase wrapping line profiles can be plotted to better evaluate the quality and amplitude of the wave. If wrapping occurs, decrease the gradient amplitude and acquire another scan.

If no adjustments to the image are necessary. Adjust parameter array size to eight equally spaced values ranging from zero seconds to a full period of the characterized resonance frequency. Acquire a scan in both the positive and negative slice orientations once the images are acquired.

Use a MATLAB program designed for generating the sheer wave data and the corresponding movie of the wave propagating in the sample. This is the file that will be needed to estimate mechanical properties. The final step of MRE is to calculate the sheer stiffness from the sheer wave images.

Begin by placing the data into the MATLAB program that will assess the three dimensional dataset, specify the imaging parameters, including field of view, gradient, amplitude, and number of bipolar pairs, and run the code. The algorithm allows the selection of regions of interest for which the mean and standard deviation of each parameter is calculated. Draw the contours of the tissue construct to select the region of interest.

The mean and standard deviation of the stiffness, storage, modulus, and loss modulus within the selected region of interest are displayed. The program also provides intermediate results, including wave after filters, wave after directional filtering and line profiles that help with estimating the faithfulness of the recovery. For an accurate estimation, the filtered wave needs to be smooth.

The standard deviation of a parameter in a specific region of interest is also an indicator of the quality of the calculation if needed, adjust other parameters as needed to get accurate values of mechanical properties to observe the changes in mechanical properties of engineered constructs as they develop. MRE testing was applied over a four week period. This construct development map shows adipogenic indicated by the letter A and osteogenic indicated by the letter O constructs with corresponding magnitude images, sheer wave images, ELAs and average shear stiffness displayed.

The color map for the ELAs corresponds with the color scheme of the bar. Chart and error bars represent the standard deviation within each constructs region of interest. Over time, the adipogenic constructs became less stiff, indicating properties that are similar to adipose tissue.

Similarly, the osteogenic constructs became more stiff over the four week period indicating bone like differentiation Once mastered, this technique can be done in about two hours if performed properly. While attempting this procedure, it's important to fully characterize the actuator Following this procedure. Methods such as biochemical analysis and histology can be used to answer questions such as the confirmation of mineral deposition.

Don't forget when working with human cellular material and MRI, it can be extremely hazardous and awareness of proper BL two and MRI. Precautions should be taken while performing these procedures.