Design and Validation of a 3D-Printed Low-Frequency Mechanical Stimulation Device for In Vitro Applications

This study describes the development, technical validation, and preliminary biological evaluation of a custom-built, low-cost device designed to apply low-amplitude mechanical vibrations to cultured cells. The system consists of 3D-printed PLA components and a miniature loudspeaker (8 Ω, 0.5 W) driven by a pulse-width modulation (PWM) module. The electrical excitation signal was verified using digital oscilloscopy and Fast Fourier Transform (FFT) analysis, which confirmed a stable fundamental frequency of 30 Hz within the mechanically effective operating range of the actuator (approximately 20–90 Hz); higher harmonic components at integer multiples of the fundamental frequency (60–180 Hz), characteristic of rectangular PWM excitation, were also detected. At the stimulation setting (30 Hz, 30% duty cycle), a MEMS accelerometer at the culture position recorded a resultant acceleration of 1.04–1.10 g (vibration-related component of approximately 0.04–0.10 g). Human gingival fibroblasts (GF06) cultured in 35 mm Petri dishes were exposed to daily vibration outside a CO2 incubator, and their response was evaluated using the MTT assay and an in vitro wound healing (scratch) assay. After 24 h of daily 15 min stimulation, the metabolic activity of vibration-treated cultures exceeded that of the controls by approximately 15 and 25 percentage points at the lower and higher seeding densities, respectively; however, these differences were not statistically significant (p = 0.189 and p = 0.098). When the daily stimulation duration was varied, the MTT signal after 72 h increased with the stimulation duration, reaching approximately 115%, 134%, and 154% of the control for 150, 300, and 450 s, respectively (p < 0.05 for 300 and 450 s). In the scratch assay, stimulation for 450 s increased gap closure compared with the control after 6 h (35.4% vs. 3.4%) and 24 h (53.4% vs. 21.2%; p < 0.01). No evident cytotoxic effects were observed under the tested conditions. The study demonstrates the technical feasibility of the platform and its ability to elicit a measurable, stimulation duration-dependent biological response, and it provides a basis for future experiments that include direct mechanical characterization of the stimulus and larger numbers of independent biological replicates under controlled physiological conditions.