Novel‐Ink‐Based Direct Ink Writing of Ti6Al4V Scaffolds with Sub‐300 µm Structural Pores for Superior Cell Proliferation and Differentiation

Abstract Ti6Al4V scaffolds with pore sizes between 300 and 600 µm are deemed suitable for bone tissue engineering. However, a significant proportion of human bone pores are smaller than 300 µm, playing a crucial role in cell proliferation, differentiation, and bone regeneration. Ti6Al4V scaffolds with these small‐sized pores are not successfully fabricated, and their cytocompatibility remains unknown. The study presents a novel ink formula specifically tailored for fabricating Ti6Al4V scaffolds featuring precise and unobstructed sub‐300 µm structural pores, achieved by investigating the rheological properties and printability of five inks containing 60–77.5 vol% Ti6Al4V powders and bisolvent binders. Ti6Al4V scaffolds with 50–600 µm pores are fabricated via direct ink writing and subjected to in vitro assays with MC3T3‐E1 and bone marrow mesenchymal stem cells. The 100 µm pore‐sized scaffolds exhibit the highest cell adhesion and proliferation capacity based on live/dead assay, FITC‐phalloidin/4',6‐diamidino‐2‐phenylindole staining, and cell count kit 8 assay. The alizarin red staining, real‐time quantitative PCR assay, and immunocytochemical staining demonstrate the superior osteogenic differentiation potential of 100 and 200 µm pore‐sized scaffolds. The importance of sub‐300 µm structrual pores is highlighted, redefining the optimal pore size for Ti6Al4V scaffolds and advancing bone tissue engineering and clinical medicine development.