Impact of Porosity and Stiffness of 3D Printed Polycaprolactone\nScaffolds on Osteogenic Differentiation of Human Mesenchymal Stromal\nCells and Activation of Dendritic Cells

Despite the potential\nof extrusion-based printing of thermoplastic\npolymers in bone tissue engineering, the inherent nonporous stiff\nnature of the printed filaments may elicit immune responses that influence\nbone regeneration. In this study, bone scaffolds made of polycaprolactone\n(PCL) filaments with different internal microporosity and stiffness\nwas 3D-printed. It was achieved by combining three fabrication techniques,\nsalt leaching and 3D printing at either low or high temperatures (LT/HT)\nwith or without nonsolvent induced phase separation (NIPS). Printing\nPCL at HT resulted in stiff scaffolds (modulus of elasticity (E):\n403 ± 19 MPa and strain: 6.6 ± 0.1%), while NIPS-based printing\nat LT produced less stiff and highly flexible scaffolds (E: 53 ±\n10 MPa and strain: 435 ± 105%). Moreover, the introduction of\nporosity by salt leaching in the printed filaments significantly changed\nthe mechanical properties and degradation rate of the scaffolds. Furthermore,\nthis study aimed to show how these variations influence proliferation\nand osteogenic differentiation of human bone marrow-derived mesenchymal\nstromal cells (hBMSC) and the maturation and activation of human monocyte-derived\ndendritic cells (Mo-DC). The cytocompatibility of the printed scaffolds\nwas confirmed by live–dead imaging, metabolic activity measurement,\nand the continuous proliferation of hBMSC over 14 days. While all\nscaffolds facilitated the expression of osteogenic markers (RUNX2\nand Collagen I) from hBMSC as detected through immunofluorescence\nstaining, the variation in porosity and stiffness notably influenced\nthe early and late mineralization. Furthermore, the flexible LT scaffolds,\nwith porosity induced by NIPS and salt leaching, stimulated Mo-DC\nto adopt a pro-inflammatory phenotype marked by a significant increase\nin the expression of IL1B and TNF genes, alongside decreased expression\nof anti-inflammatory markers, IL10 and TGF1B. Altogether, the results\nof the current study demonstrate the importance of tailoring porosity\nand stiffness of PCL scaffolds to direct their biological performance\ntoward a more immune-mediated bone healing process.