In preparation for Parylene-C coating, mechanically sheared ME materials (Metglas 2826MB-Fe₄₀Ni₃₈Mo₄B₁₈-Metglas Inc.) were cleaned and heat-treated (120 °C for 2 h) to reduce the internal stress and improve their magnetic properties. Parylene-C was then coated onto the ME materials using a Parylene deposition system (PDS 2010 LABCOTER^TM 2) following the manufacturer’s recommended protocol. Prior to implantation and in vitro culture, Parylene-C-coated ME materials were sterilized with ethylene oxide (EtO) gas. Final ME sample dimensions were 5 mm × 12.8 mm × 26 µm for all experiments.

Parylene-C (thickness of 10 µm) coated ME materials (n = 6 per group) were weighed and characterized by resonant frequency as previously described [17]. ME materials were then etched with oxygen plasma (200 mTorr) using a March Jupiter II RIE system for 0.5, 1, 3, or 5 min. ME materials were again weighed and characterized by resonant frequency following etching completion.

Surface topography measurements were made with a Nanoscope E (Digital Instruments) AFM system using constant deflection mode with a micro-fabricated silicon nitride cantilever in air. Images were processed using Digital Instruments AFM software to calculate root means squared (RMS or Rq) roughness, defined as the standard deviation of elevation with respect to the mean of a scanned measured area (5 µm²) taken over 9 sections per group. Hydrophobicity was characterized using sessile drop contact angle analysis of deionized-H₂O with a minimum of 9 measurements per sample.

To investigate the effect of Parylene-C coating on the vibration of the ME material, the resonance behavior of the material was examined. As illustrated in Figure 2, useful characteristics of the ME resonance include: (i) the resonant frequency (f₀), which is the frequency when the vibration of the material is at its highest; (ii) the resonant amplitude (S), which is the highest vibration amplitude; (iii) the resonant quality, which characterizes the spread of the resonant peak. The resonance of the ME material was captured using previously established approach [17]. Briefly, the ME materials were mechanically vibrated by exposing them to a selected frequency range of AC excitation field (160–165 kHz). In this study, ME materials coated with 5, 10, and 20 µm (n = 10 for each thickness) of Parylene-C were selected for investigation.

ME materials (n = 3 per group) coated with Parylene-C (thickness of 10 µm) were incubated in a solution of fibronectin (9.6 μg/mL in Hank’s Balanced Salt Solution, HBBS) for 45 min at room temperature, and vitronectin (0.48 μg/mL in HBSS) for 2 h at 37 °C. Resulting surface coverage of ME materials for fibronection and vitronection were 2 and 0.1 μg/cm², respectively. After rinsing the samples with HBSS, L929 fibroblasts were then seeded onto protein coated ME materials at a density 2 × 10⁴ cells/cm² and stained with a live fluorescence assay after two days of incubation at 37 °C and 5% CO₂.

For all experiments involving cell culture, L929 fibroblasts (ATCC Catalog No. CCCL-1) were maintained in standard culture medium composed of 10% FBS and 0.5% ATCC Penicillin/Streptomycin in Dulbecco’s Modified Eagles Medium (Cellgro Catalog No. 10-017-CM). Chamber slides (Lab-Tek^TM-Chamber Slide^TM system) were used to house ME materials and subsequent culture. Live/Dead assays were performed using Calcien-AM (Fluka 11783) and Ethidium Bromide (Sigma-Aldrich), respectively. Images were taken for cytotoxicity on inverted Axiovert 200 M Zeiss microscope and for adhesion on upright Olympus BX51 microscope. For vibration loading experiments, ME vibrations (Frequency 160–165 kHz) were applied to experimental set for 1 h prior to staining using previously developed in vitro ME vibrational loading system [5].

Parylene-C (thickness of 10 µm) coated ME materials (n = 3 per group) etched with oxygen plasma (0.5, 1, 3, or 5 min) were cultured with L929 fibroblasts. Cells were directly seeded on the ME materials at a density of 2 × 10⁴ cells/cm². Following incubation for 48 h at 37 °C and 5% CO₂, live/dead assays were performed using Calcien-AM (Fluka 11783) and images were obtained with an upright Olympus BX51 microscope.

In this study, a bi-lateral subcutaneous implantation model was used as a simple reproducible means to establish that the biocompatible Parylene-C coating of ME materials was viable under a dynamic in vivo host response [24,25,26]. Parylene-C coated (10 µm) and 0.5 min plasma-treated ME materials were subcutaneously implanted into the dorsal side of three age-matched male BALB/c mice (Harlan Laboratories) for 30 days using previously established implantation techniques [24]. Animals were housed and used in specific-pathogen-free facilities according to a protocol approved by the Institutional Animal Care Use Committee at Michigan Technological University. At the end of each implantation, the ME materials and surrounding tissue capsule were explanted and qualitatively assessed.

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

The additional mass loading upon deposition of Parylene-C coating is shown in Table 1. The effect of mass loading as a result of an additional Parylene-C coating on the resonance behavior of the ME material is shown in Figure 3. The application of Parylene-C coatings to ME materials dampened the vibration, and hence the secondary field response. When the Parylene-C coating thickness was increased from 0 to 20 µm, the additional mass loading reduced the maximum secondary field of ME material, A, from 0.67 to 0.33 V, a reduction of 50% on the resonant frequency. This observation is consistent with previous studies where additional weight on ME material would subsequently increase the resistance of ME vibration, hence, lowering the resonant frequency of the material. In addition, the resonant frequency and quality were reduced as a function of addition mass loading. While there was a reduction in resonant amplitude and quality, the ME materials were still able to retain their magnetostrictive properties when subjected to Parylene-C coating. The conformity of this trend also shows that the Parylene-C coating has stably integrated with the dynamic ME material substrate.

Plasma etching was shown to affect Parylene-C coating by opening and removing the benzene ring following the sequential formation of a hydroxyl then peroxy radical at the ethyl carbon site [18]. Through this processing technique, only the outer most layers of the film will possess modified surface properties [21,27]. Here, Parylene-C coated ME materials treated with different duration of plasma etching (0.5, 1, 3, and 5 min) were used to evaluate the cell reactivity to plasma-etched surface. As shown in Figure 4, fluorescent images of cells cultured on Parylene-C coated ME materials demonstrated that short plasma treatments (0.5 and 1 min) could improve the cellular adhesion.

Changes in cellular adhesion may be attributed to changes in surface properties (surface roughness and hydrophobicity) resulting from oxygen plasma treatment. As indicated in Figure 5, significantly (p < 0.01) different roughness for 0.5 and 3 min plasma-treated samples (Figure 5(A)), and significantly (p < 0.01) different hydrophobicity for all plasma-treated samples (Figure 5(B)) were evident when compared to untreated Parylene-C. However, it is interesting that similar levels of cellular adhesion occurred on samples etched for 0.5 and 1 min despite having significantly different surface roughness and hydrophobicity. Additionally, lower levels of cellular adhesion occurred on 3 and 5 min samples that possessed properties more similar to samples treated for 1 min. This result shows that plasma etching may also affect surface properties other than those directly investigated in this study, such as stiffness.

Using the ME material to monitor the effect of plasma etching on Parylene-C coated ME materials resulted in a trend of frequency increasing proportionally to etching time (R² = 0.9401, Figure 6(A)). This trend is consistent with the previously described effect of plasma etching (the removal of benzene rings corresponds to an overall change in mass), which is also consistent with the previous finding where the increase of mass decreases the resonant frequency and vice versa. The relationship of etching time and mass change is shown in Figure 6(B). As demonstrated, oxygen plasma etching technique was capable of controlling the coating mass, thus the resonant frequency, by simply altering the etching time.

The application of ME vibrations to fibroblasts cultured on functionalized Parylene-C coatings resulted in significant reductions (p < 0.01) in cell adhesion for samples adsorbed with vitronectin and plasma-treated for 0.5 min (Figure 7(A–F)). Qualitatively, noticeable changes in cell morphology (diminished cell spreading and increased roundness) were observed on samples absorbed with fibronectin, however there was no significant detachment (p < 0.0793) (Figure 7(G–I)). This outcome demonstrated the efficacy of working ME sensors in conjunction with a Parylene-C coating to modulate cell adhesion. The result also demonstrated the mechanical stability of the Parylene-C coating layer in a cell culture environment for 2 days (additional experiments showed the mechanical stability of the Parylene-C coating for a minimum of 6 months under culture conditions). Furthermore, this result showed that different functionalization techniques of Parylene-C coatings could be used to provide levels of control over the resulting cellular response to ME mechanical loading.

The application of 10 µm and 0.5 min plasma treated Parylene-C coating on ME material was shown to provide for material stability when subjected to a healthy mammalian host response. Qualitative assessment showed no signs of corrosion on explanted ME material after 30 days implantation (Figure 8). Furthermore, mice did not show any visible systemic effects to implants, exhibiting normal grooming and dietary behavior throughout the implantation period. This result demonstrated the feasibility and durability of plasma-treated and Parylene-C coated ME material within a dynamic biologic environment. Subsequent animal studies will be able to use this approach to explore in situ responses to local vibrational therapy where material stability inside a physiological environment is crucial to ensure long-term functionality of this technology.