Clinical Materials 12 (1993) 65-72

Non-Invasive Magnetic Resonance Imaging of the Soft Tissue Response to a Biomaterial

E. Khor,“* J., A. Huntp P. A. Martin,b P. J. Doherty,” R. L. Williams” & D. F. William&j a Department of Clinical Engineering, ‘Magnetic Resonance Research Centre, University of Liverpool, PO Box 147, Liverpool, L69 3BX, UK

(Received 5 December 1990; sent for revision 11 January 1991; accepted 12 November 1991)

Abstract: Magnetic resonance imaging (MRI) has been employed to visualize the tissue response to hydrogel implants in rats. High contrast MR images of the implant site were obtained. Distinct tissue variations in the MR images have been observed. These can be attributed to either the surgical procedure or the application of a tissue irritant to produce inflammation and have been verified histologically. This study demonstrates that MRI is potentially a useful tool for the non-invasive in-vivo evaluation of biomaterials.

INTRODUCTION

In-vivo evaluation is essential for any material or device being developed for clinical use. In-vivo methods require the implantation of the material or device into the target site of an animal for a predetermined time period, after which the animal is killed and the implant site recovered for the appropriate characterization. Several time periods and replicates are necessary, resulting in the use of many animals. Furthermore, the invasive and destructive nature of such procedures do not permit data to be obtained when the animal is alive. Scientific conclursions derived from such investi- gations must take this into consideration. It would clearly be desirable for a technique to be developed that will alleviate these difficulties and provide information during use, to supplement that derived post mortem.

Nuclear magnetic resonance imaging may pro- vide the means toi improve on the present situation.

* Visiting Research Fellow. Permanent address : Department of Chemistry, National University of Singapore, Kent Ridge, Singapore 05 1 I. tj TO whom correspondence should be addressed.

Since its commercial introduction in the early 198Os, magnetic resonance imaging (MRI:) has revolu- tionized the manner in which clinical diagnosis is performed. Despite the high initial cost of establish- ing an MRI facility, the technique is very attractive when compared with X-radiography, X-ray com- puter tomography and radionuclide emission tom- ography, because MRI offers no known radiation hazard to the patient. The use of MR.1 on animals has been dem0nstrated.l Polymers, for example polyvinyl alcohol,’ are routinely used in MRI phantoms. It is apparent that MRI could be extended to the non-invasive, dynamic in-vivo evaluation of biomaterial performance. The non- invasive nature of MRI could allow monitoring of the effects of an implant on live animal models over many time periods without the need for regular killing of animals. This report presents our pre- liminary results in the development of a procedure using MRI to assess the tissue response to hydrogel implants in a rat model.

Magnetic resonance imaging and image contrast

(Nuclear) Magnetic resonance imaging (MRI) is a technique that relies on a resonance phenomenon of

65 Clinical Matevials 0267-6605/93/$06.00 0 1993 Elsevier Science Publishers Ltd, England

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66 E. Khor, J. A. Hunt, P. A. Martin, P. J. Doherty, L. W~~l~~~s, D. .F Williams

the atomic nucleus. Those nuclei that possess a magnetic moment are, when placed in both a static magnetic field (B,) and an orthogonal radio fre- quency (Rfl oscillating magnetic field (B,), able to absorb energy. The oscillating Rf magnetic field is normally applied as a short duration (3.2 ms) pulse and its effect on a collection of nuclei, all absorbing energy at the same time, is to produce an overall or bulk magnetization which precesses about the external magnetic field with a resonant frequency defined according to the Lamour Equation:

n = 2pgB,

where n = the resonant frequency, g = the magneto- gyric ratio and B, is the applied static magnetic field, p = constant.

This precessing magnetic field is able to induce a small current to flow in a static conductor (the MR probe or coil) according to Maxwell’s 3rd law. This current is amplified and detected, the resulting signal being of the form of a decaying sinusoid, with a frequency depending on the magnetic field strength B,. The application of a magnetic field gradient (G,) during the reception of the signal means that the frequency of the signal will vary according to the total field strength (B, + G,). The originating nuclei effect, i.e the resonant frequency varies according to the spatial position of the originating nuclei along the magnetic field gradient G,. By extending this argument to the phase of the received signal and by the application of a series of short duration radio frequency pulses and magnetic field gradients, a series of signals can be obtained (one for each line of the finished image) which can then be processed to create an MR image. Contrast in the final image depends to some extent on the proton density differences between different tissues, but in general the tissue contrast in the final image is more greatly influenced by the effects of two relaxation times mechanisms known as TI and T,. Tl relaxation is a representation of the rate of energy loss from the system and in general this is faster for more viscous, larger molecules than for small molecules. The degree of T, induced contrast that is produced in the final image can be controlled by adjusting the ‘time for repetition’ (TR) of the Rj’ pulses. q relaxation is a representation of the rate of randomization of energy within the system and results in the loss of signal due to the effect of opposing nuclear magnetic moments cancelling each other out. In general a, relaxation is also faster for larger, more viscous, molecules but is several orders of magnitude faster than TI and its effect on

MR image contrast can be cord the time between the Rf the so-called ‘ time to echo 9 or TE

n any form of i aging, tissue contrast is not ction of the ab lute signal strength obtaine

to maximize the the tissues to be

ERIMEN

In order to estabhs non-invasive in-viv ation of biomate technique must be shown to high contrast images of t images must reveal effects caused by the im tissue or the biol Conclusions deriv quently correlate we Intramuscular i

Therefore, in our ex focused attention on material and method to give a significant tissue response which could be imaged an

is based on the presence

and fats are typical sources materials which contained sufficient mobil to be clearly visible on achieved either by inc into constEtuent molecules or by by~~at~~~. A suita choice is hydrogel, a class of polymers which include some that bihty ~ba~~~~er~st~cs~ whit amount of water and whi nuclear magnetic resonance ( ion (Ag-) solution has been inf-lammatory response in the ra Ag+ having a d’” configuration is does not interfere with the hT

the ~r~~regnati~~ of hydrogels with an aqueous

Non-invasive MRI of tissue response to a biomaterial 61

solution of Ag+ could be expected, upon leaching into the muscle, to give rise to a tissue response which can be imaged by MRI. Although new techniques are being developed for solid state imaging,3 these were not available at the time of this study and are still not sufficiently well developed to be applied to solid imaging in vivo.

MATERIALS

2-Hydrox.yethylmethacrylate (HEMA), l-vinyl pyr- rolidone l(NVP), styrene, acrylamide, benzoyl per- oxide and ethylene dimethacrylate were obtained from Aldrich Chemicals (Gillingham, UK). Stan- dard silver nitrate solution was obtained from BDH.

METHODS

Hydrogel synthesis

The copolymer hydrogel was derived from the monomers HEMA, NVP, styrene and acrylamide using a mole ratio of 4: 4: 1: 1.1 mol % (based on HEMA) of ethylene dimethacrylate and benzoyl peroxide were used as cross-linking agent and initiator, respectively. An appropriate weight of each reactant was placed in a test-tube and purged with oxygen-free nitrogen for 10 min. The mixture was then poured into PTFE moulds. These moulds were sealed and placed in an incubator at 60°C and allowed to polymerize for 3 days. The copolymer hydrogel was removed from the moulds and hydrated in distilled water. The hydrogels were soaked in sterile phosphate buffered saline (PBS) for at least 2 weeks before use. The water uptake of representative copolymer samples was found to be approximately 47 % of the gel weight.

Implant procedure

The hydrogel implant was in the form of a capsule consisting of a 6.0 mm diameter, 1 cm long hollow tube capped at both ends with fitting plugs. The lumen within the capsule was loaded with 100 ppm Ag+ solution or phosphate buffered solution (PBS) in reference samples. The loaded hydrogels were subsequently implanted bilaterally into the dorsal-, lumbar musculature of the animal model, the adult black and white hooded Lister rat of Liverpool strain. A pocket was created in the muscle and the implant was secured deep inside the muscle with a single Dexon suture. Two implants were placed in

each animal, one on either side of the midline. Each animal received a silver loaded h:ydrogel and a control (PBS loaded) hydrogel. Animals were killed at 1, 5 and 7 weeks time periods using a standard procedure.

MR imaging

MR imaging was achieved using a GE Signa (1.5 T) MR system using an in-house designed and built small animal imaging coil. This coil was an 8 strut ‘bird cage’ of length 14 cm and internal diameter 7 cm. The animal was placed inside the coil and imaging was carried out using a standard multi- echo q weighted spin warp imaging pulse sequence. TR = 500 ms, TEs = 25, 50, 75 and 100 ms, slice thickness 3 mm, pixel size 0.3 mm’, NEX = 2. The regions of inflammation were visible on all images but most clearly distinguished on the more heavily T, weighted images at TEs of 75 and 100 ms.

Histology

Following the imaging experiment, the implant and associated tissue was recovered, frozen, sectioned and stained using haematoxylin (and eosin for histological comparison.

RESULTS AND DISCUSSION

Magnetic resonance images of hydrogel implants in rats have been obtained using a small animal imaging coil. A representative coronal image of an animal after complete wound healing (2 weeks) is shown in Fig. 1. Two hydrogel implants are clearly visible within the animal. The implant on the left is seen to be partially obscured because it is not in the plane of this particular image slice.

Figures 2 and 3 show respectively, the sagittal and axial view of a rat 1 week post-implantation. The images show that the implants are surrounded by small areas of higher signal compared to the rest of the tissue. As this feature is common to both implant sites the most likely cause of this ob- servation is inflammation arising either from trauma caused by the surgical procedure, which would still be present after 1 week, or the initial reaction of the muscle to the hydrogel implant. One feature of inflammation is the local increase in water content of the tissue, resulting in a small increase in proton density and a longer T, relax- ation. This produces a stronger signal on these q weighted images. Any effects caused by the Ag+

E. Khor, 9. A. Hunt, P. A. ibfartin, P. J. Doherty, z. pKEl~~rnS, D. F.

Fig. 1. Coronal MR image of hydrogel implants in rat 2 weeks.

solution would be masked and cannot be differen- tiated at this juncture. Inflammgtion was confirmed histologically as shown in Fig. 4, indicating a promising correlation between MRI and histology.

Fig. 2. Sagittal MR image of hydrogel implants in rat i week : ieft, PBS; right, Ag-.

Figure 4(a) shows the ti loaded hydrogel, Fig. 4( loade rogel

Imaging was next performe on rats after 5 weeks

Fig. 3. Axial MR image of hydrogel implants in rat 1 week: left. PBS; right, Ag’

Non-invasive MRI of tissue response to a biomderial 69

(4

Fig. 4,. Photomicrograph showing the muscle from a rat containing hydrogels 1 week post-implant: (a) PIBS; (b) Ag+.

70 E. Khor, J. A. Hunt, P. A. Martin, P. 9. Doherty, Pp. E. Williams, D. F. Williams

Fig. 5. Axial MR image of hydrogel implants in rat, 2 days post-injection: kft, $1 right, Ag’,

post-implantation. By this time, inflammation at- tributable to the surgery would have cleared and any effects observed should be due to the Ag+ solution or hydrogel implant. No differences were found in the MR images of either implant site. Histologically, both sites were similar with very few inflammatory cells present. A possible explanation for lack of tissue inflammation could be that the Ag+ ions had precipitated, and remained inside the hydrogel structure and therefore did not influence the surrounding tissue.

In order to stimulate an inflammatory response, Ag+ solution (1 ml) was injected directly into the tissue surrounding the implant on the right side. This procedure was designed to simulate the diffusion of Ag from the hydrogel into the sur- rounding muscle tissue. At the concentration used (1 mM) the Ag+ precipitated out from normal saline (150 mM) solution. It was therefore necessary to use a solution of Ag+ in distilled water. Distilled water alone (1 ml) was used at the reference implant site. Two rats were subjected to this procedure.

Figure 5 shows an axial MR image of a rat 2 days post-injection displaying clear differences between the water and Ag+ sites. There is a distinct area of tissue damage around the Ag+ implant which appears in a different shade and can be attributed to

inflammation caused by the Ag+ solution. hotomicrograph of a tissue

Ag+ injection site confir evere inflammation. Figure 6

the response to an injection of Normal muscle tissue was observed.

Control Ag+ solution and PBS samples in agar were analyzed to determine whether the Ag’ solution or potential precipitates are i-es the lightened areas seen at the injection 7 shows four images of agar susp that there are no contrast di Ag+ solution (a), the Ag+ preci respective references, water (c) a contrast differences in the MR images from~ the rat must therefore be attri inflammation caused by the Ag+ solution an artefact generated by the presence of Ag+ in the tissue.

lusion, the preliminary work wn has the ability to detect tissue s if reaction arises from t e presence of an

implant, This result is e~c~u~~~i~~ and demon strates that MRI could be develo as a useM tool for the in-vivo ~va~~atio~ of interactions

Non-invasive MRI of tissue response to a biomaterial 71

Fig. 6. Photomicrograph showing the muscle from a rat containing hydrogels, 2 days post-injection: (a) F’BS; (b) A Lg+

72 E. Khor, J. A. Hunt, P. A. Martin, P. J. Doherty, Pz. L. Uiams, D. IT

Fig. 7. MR images of Ag+ solutions in agar. (a) Ag+ in water; (b) Ag+ in P S; (c) water in agar; (d) PBS in agar.

ACKNOWLEDGEMENTS

E.K. would like to thank the Association of Commonwealth Universities for the award of a Development Fellowship. J.A.H. would like to thank the Science and Engineering Research Coun- cil for a research studentship.

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4.

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