 The
mission of the CIA-MR is to
apply magnetic resonance technology to fundamental problems in
engineering and the physical sciences.
CIAMR HomeCIAMR PersonnelCIAMR FacilitiesMR-PET Scanner DevelopmentMagnetic LevitationMR Compatibility of Medical DevicesMR Analysis of Bioreactor Structure and FunctionIndustrial NMR ApplicationsMRI of Wood3D MRI of FoamTissue EngineeringMRI of KneesMicro NMR CoilsDepartment of RadiologyUniversity of Minnesota |
Tissue Engineering CIA-MR Transport of DMSO into Artificial Dermis   ^ MOVIE ^ Use of a combined C4S-Keyhole imaging technique to study the dynamics of cryoprotective agents in an engineered tissue (Presented at ISMRM 98, Sydney, Australia, April 1998) N.P. Bidault(1,2), B.E. Hammer(1,2), A. Hubel(1) (1) Biomedical Engineering Center, (2) CIA-MR, University of Minnesota, Minneapolis, MN55455, USA Introduction Understanding the dynamics of cryoprotective agents (CPA) in tissues is of great importance in the design of optimal cryopreservation protocols. Spectroscopic techniques have been used (1), however, there is a need to obtain spatial information on the dynamics of cryopreservation. The C4S (2) imaging technique has been used to selectively study the permeation of dimethyl sulfoxide (DMSO) into rat kidney and rat liver slices (3). However, the inherently long imaging time necessary to obtain good signal-to-noise ratio limits the C4S technique to slow processes which highly depend on the tissue type and CPA. Some studies have indicated that the dynamics of glycerol into split-thickness skin is quite fast (4). Engineered skin tissues have lower cell densities than natural tissues and therefore will experience faster transport relative to indigenous tissue. This was confirmed in our laboratory. Hence, to study the dynamics into an artificial dermis we have decided to accelerate the C4S imaging technique by combining it with the keyhole (5) technique. Materials and method 0.5%w/v bovine corium collagen (Kensey-Nash, Exton, PA) was mixed with 1:19w/w hyaluronic acid (Lifecore Biomedical, inc., Chaska, MN) and dispersed in HCl at pH=3.0. The dispersion was frozen for 24 hours, then freeze-dried for 3 days. The resulting collagen sponge was crosslinked in a vacuum oven for 5 days. Fibroblasts (Clonetics, inc., San Diego, CA) were grown in a cell factory (Nunc, inc., Naperville, IL) for 5 days. The medium, composed of DMEM supplemented with 10% FBS, 10,000 units/ml penicillin G and 10,000 mg/ml streptomycin and 10%w/w ascorbic acid, was changed every two days. The harvested fibroblasts were introduced on the surface of the sponge at a concentration of 9.105 cells/cm2. After 14 days of culture in an incubator at 37° C and 5% CO2, the artificial dermis was placed in a homemade plastic holder and introduced in a 10mm glass tube filled with Hank’s Basic Salt Solution (HBSS). The tube was then introduced in a 20 mm homemade birdcage coil. The solution was removed and replaced by the CPA solution composed of HBSS and 50% DMSO as quickly as possible using two Masterflex (Cole-Parmer, Chicago, IL) pumps to avoid dehydration of the engineered tissue. The experiment was carried out at room temperature. All imaging was carried out on a 200 MHz 21-cm horizontal bore magnet (Magnex Scientific Limited, Oxon, UK) and Techmag (Techmag, Houston, TX) imaging accessory. The C4S pulse sequence has been described elsewhere (2). Parameters used to acquire the images were as follows: chemical shift between water and DMSO peaks in the proton spectrum at 5T: 440 Hz, pulse bandwidth, 720 Hz; sweep-width, 3000 Hz, in-plane gradient strength, 1.2 G/cm; slice thickness, 2 mm; in-plane resolution, 80 mm; TE= 30 ms; TR= 1 s; number of scans, 2; two step phase cycling, 128 time domain points. The carrier frequency was set at the DMSO frequency. Time t=0 corresponds to the introduction of the CPA solution. At t=0, the dynamics C4S-keyhole images were started using 32 phase encode steps. The time to acquire the C4S-keyhole images was 1 min. After equilibration of the tissue, a 128´ 128 image was acquired to reconstruct the keyhole images using IDL software (RSI, Boulder, CO). The images were then stacked into a movie format. Results Shown below are four C4S-keyhole images of the dynamics of DMSO into an engineered dermis. Figure 1 corresponds to the first image of the series started at t=2min. Figure 1-4 show four images obtained ten minutes apart from each other.  Fig. 1 DMSO image at t=2 min
............Fig. 2 t=12 min
 Fig. 3 t=22
min.....................................Fig. 4 t=32 min
The cell density in the tissue is about 2.8 104 cells/mm3 and the cells are homogeneously distributed in the tissue (unpublished results). Given the in-plane resolution and the slice thickness, the voxel size is 12.8 10-3 mm3. Therefore each voxel corresponds to about 350 cells. Conclusion The use of the combined C4S-keyhole imaging technique is well appropriate for the spatial and time resolutions in the study of dynamics of CPAs in engineered tissues. We are currently working on the development of a mathematical model to describe these dynamics. The series of images will be used to solve the model. References Walcerz, D.B., et al., Cell Biophysics 26, 79-102, 1995. Volk, A., et al., J. Magn. Reson., 71, 168-174, 1987. Isbell, S. A., In press. Zieger, M.A.J., et al., Cryobiology, 35, 53-69, 1997. van Vaals, J.J., et al., JMRI, 3(4), 671-5, 1993. |
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