Optimization and validation of a test bench and protocols for characterizing the permeability of hard and soft tissue engineering scaffolds

Tissue engineering (TE) strategies are based on the active interplay among cells, three-dimensional (3D) scaffolds, and physiological signals in view of developing in vitro functional tissue substitutes. Scaffold effectiveness is strongly influenced by its microstructure and ability to be permeated by fluids (i.e., scaffold permeability) and consequently to be colonized by cells. Permeability depends on the combination of porosity, pore size, tortuosity, and interconnectivity and its characterization is crucial for an effective evaluation of the overall scaffold performance. Several methods and set-ups have been proposed to characterize the permeability of hard or soft materials, but no systems able to characterize both types of materials have yet been developed. Inspired by this context, we developed a versatile permeability test bench (PTB), based on the pump method, for characterizing hard and soft scaffolds. The permeability chamber (PC), designed using the Solidworks software and manufactured by stereolithography, consists of two parts coupled by screws, with an internal cylindrical geometry. Interchangeable silicone holders and a grid allow housing hard or soft cylindrical samples (height = 1-14 mm, diameter = 8-27 mm). The PC is part of a closed loop hydraulic circuit based on a peristaltic pump, a reservoir for demineralized water, silicone tubing, 3-way stopcocks, and two in-line relative pressure sensors located upstream and downstream the PC, respectively, to measure the pressure drop across it. The sensor signals are acquired by a DAQ, controlled by a computer running a purpose-built LabView interface. Upon imposing a defined flow rate (selected for guaranteeing laminar flow), scaffold permeability (k) was calculated by using the Darcy flow transport model and considering the pressure drop across the sample (subtracting the mean pressure drop due to the empty chamber). For validating the PTB, two types of scaffolds for bone TE were tested: commercial biomimetic scaffolds (SmartBone) and 3D-printed PLA scaffolds with different geometries. For each sample, 4-6 tests were carried out (flow rate = 5 ml/min), and for each test 5 pressure recordings were performed. As reference test bench (RTB), a previously validated device based on the acoustic method and developed by the National Institute of Metrological Research (INRiM) was used. The mean permeability values of the SmartBone scaffolds were 2.3·10-10 ± 3.4·10-10 m2 (PTB) and 1.8·10-10 ± 4.2·10-11 m2 (RTB), while the mean permeability values of the PLA scaffolds tested within the PTB and the RTB resulted to be respectively: 4.1·10-10 ± 3.1·10-10 m2 and 4.0·10-10 ± 5.5·10-11 m2 for perpendicular geometry, 2.4·10-10 ± 1.7·10-10 m2 and 2.8·10-10 ± 5.2·10-11 m2 for oblique geometry, 2.4·10-10 ± 1.4·10-10 m2 and 2.2·10-10 ± 4.0·10-11 m2 for random geometry. Although the PTB measurements were affected by a higher dispersion, the normalized errors between the results obtained with the PTB and RTB resulted to be less than 1, confirming the suitability of the PTB as permeability test bench for hard scaffolds. For reducing measurement uncertainty, the optimization of the PTB data acquisition system is in process. In parallel, measurements with a USB digital differential pressure transducer, selected for reducing the transient phenomena, are ongoing together with further tests varying the flow rates. Finally, tests are currently underway for assessing the suitability of the PTB to be used for soft scaffolds characterization.