A bioreactor for controlled electromechanical stimulation of developing scaffold-free constructs

ABSTRACT Bioreactors are commonly used to apply biophysically-relevant stimulations to tissue-engineered constructs in order to explore how these stimuli influence tissue development, healing, and homeostasis. These bioreactors offer great flexibility as key features of the stimuli ( e . g ., duty cycle, frequency, amplitude, duration) can be controlled to elicit a desired cellular response. Controlled delivery of mechanical and/or electrical stimulation has been shown to improve the structure and function of engineered tissue constructs, compared to unstimulated controls, for applications in various musculoskeletal soft tissues. However, most bioreactors that apply mechanical and electrical stimulations, do so to a scaffold after the construct has developed, preventing study of the influence these stimuli have on early construct development. Thus, there is a need for a bioreactor that allows the direct application of mechanical and electrical stimulation to constructs as they develop , to enable such exploration and to better mimic key aspects of tissue development. Hence, the objective of this study was to develop and calibrate a bioreactor to deliver precise mechanical and electrical stimulation, either independently or in combination, to developing scaffold-free tissue constructs. Standard Flexcell Tissue Train plates were modified with stainless steel loading pins and stimulating electrodes to integrate direct mechanical and electrical stimulation, respectively, into our established scaffold-free, single-fiber engineering platform. Linear calibration curves were established, then used to apply precise dynamic mechanical and electrical stimulations, over a range of physiologically relevant strains (0.50, 0.70, 0.75, 1.0, 1.5%) and voltages (1.5, 3.5 V), respectively. Once calibrated, applied mechanical and electrical stimulations were not statistically different from their desired target values, and were consistent whether delivered independently or in combination. Indeed, concurrent delivery of mechanical and electrical stimulation resulted in a negligible change in mechanical (< 2%) and electrical (<1%) values, from their independently-delivered values. With this calibrated bioreactor, we can apply precise, controlled, reproducible mechanical and electrical stimulations, alone or in combination, to scaffold-free, tissue engineered constructs as they develop, to explore how these stimuli can be leveraged to advance and accelerate functional tissue engineering in a variety of musculoskeletal soft tissues. IMPACT STATEMENT As the importance of biophysical stimulation in tissue engineering continues to be recognized and incorporated, this bioreactor provides a platform to further our understanding of the roles independent or combined mechanical and electrical stimulation have in tissue development and functional maturation, and may inform future tissue engineering approaches for clinical applications.