A commercial magnetoelastic material, Metglas 2826MB (Fe₄₀Ni₃₈Mo₄B₁₈) from Metglas, Inc., was used to create ME films for both experiments. The material was a 12.7 mm wide ribbon with a thickness of 28 mm. For the first task, the ME films were rectangles created by mechanically shearing the Metglas ribbon into strips with dimensions of 12.7 mm × 5 mm. The strips were sonicated in isopropanol for 5 minutes and annealed for 2 hours at 125˚C to remove internal stress. For the second task, two rectangular conjoined strips of 12.3 mm × 2.7 mm and 13.7 mm × 2.7 mm were created. The strips were separated by a gap of 1.0 mm via a bridge of 0.5 mm wide (see Figure 1). The conjoined ME strips were created with an etching process. First, both sides of the as-purchased ME ribbon were coated with Nazdar^® 59,000 Series Enamel Plus Gloss Screen Ink 59,192. After the ink dried, the outline of the conjoined strips was carved on one of the painted surface with a CNC Microplotter Mill, exposing the ME material beneath. The patterned ME ribbon was then immersed in a ferric chloride etching bath for 20 - 30 minutes at 45˚C to etch out of the conjoined strips. The ink was removed from the strips by rubbing with acetone and the conjoined strips were then sonicated and annealed as previously described. Parylene-C was coated onto the ME films for both experiments using a Parylene deposition system (PDS 2010 LABCOTER^TM 2) with the manufacturer’s recommended protocol. The films were then etched with oxygen plasma (200 mTorr) using a March Jupiter II RIE system to create a surface conducive to cell adhesion. All ME films were sterilized with ethylene oxide gas before cell culture.

The ME films were vibrated independently using a customized system previously developed [8-10]. The ME films were placed in a chamber slide within a magnetic coil. A custom, microcontroller-based ME interrogation

system was used to generate the excitation magnetic fields. The resonant behavior of the ME films were determined by measuring their magnetic field response at varying frequencies.

L929 fibroblasts (ATCC CCCL-1) were used in all experiments involving cell culture. The cells were maintained in standard culture medium composed of 10% fetal bovine serum (FBS) and 0.5% ATCC Penicillin/ Streptomycin in Dulbecco’s Modified Eagles Medium (Cellgro Catalog No. 10-017-CM). Chamber slides (LabTek^TM-Chamber Slide^TM system) were used to hold the ME films and cell cultures. Calcien-AM (Fluka 11783) was used to fluorescently label live cells adhered to the ME substrate surface. Images were taken with an upright Olympus BX51 microscope, and cellular adhesion was determined by direct cell counting.

The rectangular ME films used in the first task were placed in the chamber slide, followed by cells seeded at a density of 1 × 10⁴ cells/cm². These ME films were separated into two groups: control (no vibrational loading), and vibrational loading at 171 - 176 kHz which was at least 10 kHz higher than the resonant frequency of the strip. After 48 hours of incubation at 37˚C and 5% CO₂, ME vibrations were applied to the ME film for 1 hour followed by staining with Calcien-AM.

For the second task, the two conjoined ME strips were separated into three groups: control (no vibrational loading); vibrational loading at 171 - 176 kHz, and vibrational loading at 152 - 157 kHz. The experimental process was identical to that previously described for the first task.

Statistical analyses were made using standard student’s t-test or analysis of variance (ANOVA) on JMP software. All experiments were conducted in triplicate, unless otherwise stated. Data is expressed as the mean ± S.E.M. (standard error of the mean) and p-values less than 0.05 (p < 0.05) were considered significant.

The resonance of the single strip ME film is plotted in Figure 2. The resonant frequency of the strip was about 160 kHz. Figure 2 also indicates the frequency range of excitation field applied to the single strip ME films, which was at least 10 kHz higher than the resonant frequency of these strips. The resonance of the two-strip ME film is shown in Figure 3. As each ME strip has a distinct resonant frequency, two resonant peaks were ob-

served. The excitation frequencies applied to these films are also indicated.

Adhesion of L929 fibroblasts to the ME films can be controlled by changing the vibration magnitude, which can be easily achieved by changing the frequency of the excitation field (see Figure 3). As shown in Figure 4 A)-F), qualitative images of fibroblast adhesion indicated significant differences between controls and vibrated groups (at resonant frequency and off-resonant frequency) when quantified. Compared to the controls (no vibration), ME strips that were vibrated at the resonant frequency showed a significant decrease. On the other hand, ME strips that were vibrated at off resonant frequency showed an increase in cell adhesion when compared to the controls.

As indicated in Figure 5, ME films that were vibrated at the resonant frequency showed significant reduction in cell adhesion (p < 0.05) while films that were vibrated at frequencies away from the resonance showed an increase in cell adhesion. These quantitative results demonstrate that the larger vibrations (0.1 - 0.15 µm) inhibited cell adhesion. However, at low vibration levels (<10 nm), a result of excitation by magnetic fields of frequencies at least 10 kHz from the resonant frequency, cell adhesion increased. Therefore, these results suggest that ME materials have the potential to create a surface that can be precisely tuned for increasing or decreasing cell adhesion for specific applications.

Results from Figures 4 and 5 indicate that under certain conditions, vibrations can also increase cell adhesion. This behavior was not reported in our previous work that focused on vibrational strains at 0.1 - 0.15 µm. To ensure the increase in cell adhesion is not caused by the interference from the neighboring vibrating strip, single strip ME films (task 1) were used to load cells with off frequency vibrations. Similar to the observations on 2-stip ME films, qualitative results from the single-strip ME films indicated that application of very small vibrations at off resonant frequency increased fibroblast adhesion compared to static controls. This observation is consistent with other studies, in which nanometer vibrations also showed an increase in fibroblast adhesion [11]. Quantitative results indicated that off-resonant frequency vibrations significantly increase (p < 0.05) fibroblast

adhesion to ME materials, as illustrated in Figures 6 and 7. These results demonstrate that ME materials can be designed to increase or decrease fibroblast cell adhesion depending on desired cell attachment response.