Development and characterization of a bioreactor stimulation unit providing cyclic hydrostatic pressure for bone tissue engineering

Fluid flow-induced forces play a key role in fluid-filled tissues. In bone tissue, the external mechanical loading is transmitted to the cells by the movement of the interstitial fluid in the lacuna-canalicular system and by changes in the bone marrow pressure in trabecular bone. These forces influences bone cell fate, ultimately regulating skeletal adaptation to mechanical loading. In vitro studies have demonstrated that the application of native-like mechanical stimuli, such as fluid flow-induced shear stress and hydrostatic pressure, enhance bone cell proliferation and differentiation, although further research is needed to unravel the effects of cyclic pressure on bone tissue models. In this perspective, bioreactors represent a key technology for in vitro development and investigation of functional bone tissue models under defined and controlled stimuli. In this context, the aim of this thesis was to design, optimize, characterize, and validate a versatile physical stimulation unit, to be used within a perfusion bioreactor previously developed, which implements the hydrostatic pressure (HP) stimulus for mimicking the native bone environment. In detail, the key component of the stimulation unit is a pinch valve connected to the perfusion circuit and located downstream the bioreactor culture chamber. The pinch valve, controlled by a control unit based on an Arduino microcontroller and a user-friendly interface, is cyclically activated for inducing intermittent increasing pressure inside the culture chamber. The stimulation unit has been designed to provide cyclic compression to the construct by controlling the pinch valve in terms of stimulation period (100-5000 ms) and duty cycle (1-100 %). Moreover, a monitoring unit based on a microprocessor (Beagle Bone Black) was designed and programmed. Subsequently, testing, optimization, characterization, and validation of the stimulation unit were carried out. A test bench, based on non-invasive pressure transducers (SP 844) connected to a DAQ (National Instruments) controlled via a LabVIEW interface (National Instruments), was developed for characterizing the pressure evolution within the culture chamber. The stimulation unit performance was assessed by measuring the pressure developing within the culture chamber imposing different parameter configurations, including flow rate (0.3-1 mL/min), stimulation period (1-5 s), and duty cycle (25-50-75 %), and postprocessing the signal with Matlab (MathWorks). Imposing the different configurations, pressure values ranging from 2 to 35 kPa were obtained, which are comparable with intramedullary pressure values recorded in physiological conditions in bone marrow and which were demonstrated to drive the osteogenic lineage commitment of human bone marrow stem cells. In parallel, it was demonstrated that the monitoring unit allows data from the pressure transducer to be received, processed, displayed, and stored. In the future, the monitoring and control units will be combined to provide fully automated real-time monitoring and adaptive stimulation.