Development and testing of a microcontroller-based system for the electrical stimulation of engineered skeletal muscle tissues

Skeletal muscle tissue engineering aims to develop functional muscle constructs in vitro for applications in regenerative medicine, drug testing, and in vitro disease modeling. Efficient maturation of engineered muscles relies on the precise recapitulation of the native biophysical stimuli. In vivo, electrical pulses are essential for coordinated contraction of the motor units. In vitro, the ability to deliver defined patterns of stimulation is essential to induce a contractile behaviour in the cultured constructs, promote cell differentiation, and replicate a physiologic-like activation, in the form of single twitches or of tetanic contractions. In this thesis, a novel electrical stimulation system for skeletal muscle tissue engineering was developed and tested. The new device was developed by upgrading the waveform generation capability of a previously developed electrical stimulator for cardiac tissue engineering (ELETTRA). The existing ELETTRA allowed to deliver electrical mono/biphasic pulses with amplitude 0.25-12 V, frequency (0.5-10.0 Hz) and duration (1-10 ms), suitable for cardiac tissue engineering. In this work, the control software was re-designed to comply with the specific needs of skeletal muscle tissue engineering extending the frequency range between 0.5 and 140 Hz, adapting the pulse duration range (1 to 6 ms). The key innovation was the exploitation of the Arduino Due internal timer-counters to make the three stimulation channels independent. This software-oriented design allowed parallel and efficient generation of electrical stimuli, enabling the possibility to apply new stimulation patterns. In addition to the original single-pulse mode, two new stimulation modes were introduced: - Train mode , to deliver trains of pulses with period (200 -1500 ms) and duty cycle (20-80%) control . - Square mode, to generate constant symmetric square waveforms in the same range of frequency of the single asimmetric pulses. These additions improved the device's ability to simulate physiological activity. Moreover, the new frequency range and the train mode enable compatibility with stimulation protocols capable of causing tetanic contraction, in accordance with literature data. To evaluate system performance, a two-phase testing method was employed. Firstly, a simplified circuit with LEDs emulated output stages, allowing real-time debugging and verification of timing logic. In the second phase, a breadboard realization of a full output channel of the stimulator was made, and direct acquisition and characterization of the waveforms supplied to a digital oscilloscope were performed. Data were collected and analysed in MATLAB to assess the accuracy, replicability, and conformity of the waveforms to the set technical specifications. Acceptance tests confirmed compliance with the set requirements, and a novel prototype of the stimulator, named ELETTRA 4.0, was built for use by biological laboratory operators. In conclusion, the developed stimulator delivers a controlled and stable electrical stimulation suitable for skeletal muscle tissue engineering, while maintaining all the features suitable for cardiac tissue engineering. ELETTRA 4.0 provides control accuracy, versatility, parallelization and portability at a competitive production cost (< 400 €), representing a powerful tool for investigating the influence of the electrical stimulation on muscle development and disease.