1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 2Department of Engineering Mechanics, University of Nebraska-Lincoln
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Curtis, E. T., Zhang, S., Khalilzad-Sharghi, V., Boulet, T., Othman, S. F. Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth. J. Vis. Exp. (60), e3618, doi:10.3791/3618 (2012).
Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (μMRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, μMRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2>0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.
1. Tissue Construct Preparation
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. The procedure for construct preparation is based on methods conducted by Dennis et al., Hong et al., and Marion and Mao8,9,10.
2. Actuator Characterization
Characterization of the actuator is a vital step for the MRE experiment. MRE 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 piezoelectric actuator. An illustrated example of the characterization process is shown in Figure 3. The goal of this procedure is to optimize the motion of the actuator in order to generate harmless shear waves with significant amplitudes (~250 micron).
3. Image Acquisition
4. MRE Experiment Image Processing
Note: By assuming a planar shear wave, the equations of motion decouple allowing the estimation of the complex-valued shear modulus as a function of the displacement and its Laplacian. The algorithm approximates spatial second derivatives with finite difference and computes the shear modulus on a pixel-by-pixel basis. From this complex number, many mechanical parameters can be deduced such as the shear wave speed, wave attenuation, shear stiffness, shear elasticity, shear viscosity, etc. The algorithm also allows the selection of regions of interest for which the mean and standard deviation of each parameter is calculated.
Note: The program provides intermediate results (wave after low-pass filters, wave after directional filtering, temporal FFT, line profiles, etc.) that help the user estimate the faithfulness of the recovery.
5. Representative Results
Figure 4 notes the change in mechanical properties throughout four weeks of osteogenic and adipogenic construct development. MRE was conducted at 730-820 Hz. While both seeded sponges started at approximately 3 kPa, osteogenic directed tissues resulted in a stiffness of 22 kPa; whereas, adipose directed tissues decreased in stiffness to 1 kPa. Furthermore, the osteogenic constructs showed a notable decrease in size in comparison from beginning to end of the study. Additional properties derived from elastography study are shown in Table 1.
Figure 1. The image acquisition process for magnetic resonance elastography. During image acquisition, a pulse sequence (a) controls the synchronization (b) of the function generator with the bipolar gradients pulses of the MRI scanner. Following acquisition of bipolar gradients toggled in positive and negative orientations, (c) a shear wave image is produced using complex division.
Figure 2. Flow diagram of the MRE process for tissue engineered constructs. First, cells (a) are first grown and expanded to the population size essential for the designed project. Then the cells are seeded (b) onto a biomaterial scaffold and chemical reagents are applied to signal differentiation. Scaffolds are characterized with MRE, whose first step (c) is the determination of the resonance frequency of the actuator coupled to the construct. Next, MRI images (d) are acquired to generate a shear wave image (e). Finally, an algorithm is applied to yield an elastogram (f) that maps the stiffness of the construct. Concurrently, constructs are sectioned for histological assessment (g) in order to validate differentiation.
Figure 3. Actuator characterization procedure. The gelatin scaffold is enclosed by a 0.5% agarose gel. To characterize the motion being transferred into the sample a white noise is first sent into the system (1a) and the resulting motion is detected using a Laser Doppler Vibrometer (1b). Once the resonance frequency is determined, a continuous sinusoid signal at resonance (2a) is sent to determine the displacement (2b) transferred to the gelatin environment.
Figure 4. Construct development map over four week period. Adipogenic (A) and osteogenic (O) constructs are shown from left to right with corresponding magnitude and shear wave images, elastogram, and average shear stiffness. The colormap for the elastogram corresponds with the color scheme of the bar chart and error bars represent the standard deviation within each construct's region of interest.
Table 1. Mechanical properties of adipose and osteo constructs over a four week period of growth.
In this procedure, the process of MRE for tissue engineered constructs is demonstrated from cell preparation to the generation of an elastogram. By applying a nondestructive mechanical assessment method to the tissue engineering pipeline, it is now possible to evaluate changes in engineered constructs throughout multiple stages of development. In addition, MRE complements other MR methods for monitoring tissue engineered constructs such as diffusion, magnetization transfer, and chemical shift analysis1.
When performing MRE experiments, a few limitations should be noted. The assessment of in vitro specimens is a time sensitive study. Therefore, it is recommended that studies should last no more than one hour so that any potential damage to the tissue construct is minimized. Additionally, faithful recovery of the stiffness map can be compromised due to constructs being either too small or stiff 6. One possible solution to this problem is to operate at higher frequency (> 2.5 kHz), as the wavelength is inversely proportional to the frequency. Piezoelectric stack actuators driven by high voltage amplifiers are able to deliver sufficient motion at such frequencies to produce a full shear wavelength in the sample. Another possible amendment to the protocol is to use faster sequences such as fast spin-echo and echo planar imaging11, 12.
Beyond the possibilities of MRE for tissue engineered constructs in vitro, the next step of pre-clinical assessment is to evaluate the development of tissues implanted into a living system. The application of MRE to mouse studies would provide another opportunity to nondestructively evaluate the development the tissue constructs. Extension of elastography for treatment of bone or cartilage defects would potentially provide a better understanding of how to produce longer lasting functional implants for use in regenerative medicine. Magnetic resonance elastography has the potential to play an increasing role in the validation of engineered constructs both in vitro and in vivo.
The authors have no conflicts of interest to disclose.
This research was supported in part by NIH RO3-EB007299-02 and NSF EPSCoR First Award.
|MSCGM-Bullet Kit||Reagent||Lonza Inc.||PT-3001||Store at 4°C|
|0.05% Trypsin-EDTA||Reagent||GIBCO, by Life Technologies||25300-054||Store at -20°C|
|3-Isobutyl-1-methylxanthine||Reagent||Sigma-Aldrich||I5879||Store at -20°C|
|Insulin-bovine pancreas||Reagent||Sigma-Aldrich||I6634||Store at -20°C|
|L-Ascorbic Acid 2-phosphate||Reagent||Sigma-Aldrich||A8960|
|Gelfoam||Scaffold||Pharmacia Corporation (Pfizer)||09-0315-08|
|Human mesenchymal stem cells||Cell Line||Lonza Inc.||PT-2501|
|9.4T MR Scanner||Equipment||Agilent Technologies||400MHz WB|
|10mm Litz Coil||Equipment||Doty Scientific|
|Laser Doppler Vibrometer||Equipment||Polytec||PDV-100|
|Function generator||Equipment||Agilent Technologies||AFG 3022B|
|Piezo Bending motor||Equipment||Piezo Inc.||T234-A4Cl-203X|
|Computer-Linux||Equipment||Intel||Processor: Intel Core 2 Duo E8400, Memory: 2G|
|Computer-Windows||Equipment||Intel||Processor: Intel Core 2 Duo E8400, Memory: 2G|