Unraveling the transcriptome profile of pulsed electromagnetic field stimulation in bone regeneration using a bioreactor-based investigation platform.

INTRODUCTION: Human mesenchymal stem cells (hMSCs) sense and respond to biomechanical and biophysical stimuli, yet the involved signaling pathways are not fully identified. The clinical application of biophysical stimulation including pulsed electromagnetic field (PEMF) has gained momentum in musculoskeletal disorders and bone tissue engineering. METHODOLOGY: We herein aim to explore the role of PEMF stimulation in bone regeneration by developing trabecular bone-like tissues, and then, culturing them under bone-like mechanical stimulation in an automated perfusion bioreactor combined with a custom-made PEMF stimulator. After selecting the optimal cell seeding and culture conditions for inspecting the effects of PEMF on hMSCs, transcriptomic studies were performed on cells cultured under direct perfusion with and without PEMF stimulation. RESULTS: We were able to identify a set of signaling pathways and upstream regulators associated with PEMF stimulation and to distinguish those linked to bone regeneration. Our findings suggest that PEMF induces the immune potential of hMSCs by activating and inhibiting various immune-related pathways, such as macrophage classical activation and MSP-RON signaling in macrophages, respectively, while promoting angiogenesis and osteogenesis, which mimics the dynamic interplay of biological processes during bone healing. CONCLUSIONS: Overall, the adopted bioreactor-based investigation platform can be used to investigate the impact of PEMF stimulation on bone regeneration.

Adaptable test bench for ASTM-compliant permeability measurement of porous scaffolds for tissue engineering.

Intrinsic permeability describes the ability of a porous medium to be penetrated by a fluid. Considering porous scaffolds for tissue engineering (TE) applications, this macroscopic variable can strongly influence the transport of oxygen and nutrients, the cell seeding process, and the transmission of fluid forces to the cells, playing a crucial role in determining scaffold efficacy. Thus, accurately measuring the permeability of porous scaffolds could represent an essential step in their optimization process. In literature, several methods have been proposed to characterize scaffold permeability. Most of the currently adopted approaches to assess permeability limit their applicability to specific scaffold structures, hampering protocols standardization, and ultimately leading to incomparable results among different laboratories. The content of novelty of this study is in the proposal of an adaptable test bench and in defining a specific testing protocol, compliant with the ASTM International F2952-22 guidelines, for reliable and repeatable measurements of the intrinsic permeability of TE porous scaffolds. The developed permeability test bench (PTB) exploits the pump-based method, and it is composed of a modular permeability chamber integrated within a closed-loop hydraulic circuit, which includes a peristaltic pump and pressure sensors, recirculating demineralized water. A specific testing protocol was defined for characterizing the pressure drop associated with the scaffold under test, while minimizing the effects of uncertainty sources. To assess the operational capabilities and performance of the proposed test bench, permeability measurements were conducted on PLA scaffolds with regular (PS) and random (RS) micro-architecture and on commercial bovine bone matrix-derived scaffolds (CS) for bone TE. To validate the proposed approach, the scaffolds were as well characterized using an alternative test bench (ATB) based on acoustic measurements, implementing a blind randomized testing procedure. The consistency of the permeability values measured using both the test benches demonstrated the reliability of the proposed approach. A further validation of the PTB's measurement reliability was provided by the agreement between the measured permeability values of the PS scaffolds and the theory-based predicted permeability value. Once validated the proposed PTB, the performed measurements allowed the investigation of the scaffolds' transport properties. Samples with the same structure (guaranteed by the fused-deposition modeling technique) were characterized by similar permeability values, and CS and RS scaffolds showed permeability values in agreement with the values reported in the literature for bovine trabecular bone. In conclusion, the developed PTB and the proposed testing protocol allow the characterization of the intrinsic permeability of porous scaffolds of different types and dimensions under controlled flow regimes, representing a powerful tool in view of providing a reliable and repeatable framework for characterizing and optimizing scaffolds for TE applications.

Tailoring the Microarchitectures of 3D Printed Bone-like Scaffolds for Tissue Engineering Applications.

Material extrusion (MEX), commonly referred to as fused deposition modeling (FDM) or fused filament fabrication (FFF), is a versatile and cost-effective technique to fabricate suitable scaffolds for tissue engineering. Driven by a computer-aided design input, specific patterns can be easily collected in an extremely reproducible and repeatable process. Referring to possible skeletal affections, 3D-printed scaffolds can support tissue regeneration of large bone defects with complex geometries, an open major clinical challenge. In this study, polylactic acid scaffolds were printed resembling trabecular bone microarchitecture in order to deal with morphologically biomimetic features to potentially enhance the biological outcome. Three models with different pore sizes (i.e., 500, 600, and 700 µm) were prepared and evaluated by means of micro-computed tomography. The biological assessment was carried out seeding SAOS-2 cells, a bone-like cell model, on the scaffolds, which showed excellent biocompatibility, bioactivity, and osteoinductivity. The model with larger pores, characterized by improved osteoconductive properties and protein adsorption rate, was further investigated as a potential platform for bone-tissue engineering, evaluating the paracrine activity of human mesenchymal stem cells. The reported findings demonstrate that the designed microarchitecture, better mimicking the natural bone extracellular matrix, favors a greater bioactivity and can be thus regarded as an interesting option for bone-tissue engineering.