EXPECT: A thermosensitive embedded bioprinting platform for guided spatial cell organization

Complex tissue engineering requires precise spatial cell organization, but static or isotropic hydrogels hinder long-term pattern maintenance due to random cell migration. We developed EXtrusion Patterned Embedded ConstruCT (EXPECT), a thermosensitive hydrogel embedding medium for 3D bioprinting, integrating Carbopol® 940 and gelatin for rheological properties and print fidelity, with poly (N-isopropylacrylamide)-graft-chondroitin sulfate (pNIPAAm-CS) for biocompatibility and temperature-responsive behavior (~32 ?°C lower critical solution temperature (LCST)). Rheological and small-angle X-ray scattering (SAXS) analyses confirmed EXPECT's self-healing printability and reversible LCST-driven transitions from hydrophobic (above ~32 ?°C) to hydrophilic (below ~32 ?°C) states. Temperature actuation (15 ?min at 25 ?°C every ~5 days, otherwise 37 ?°C) in 10 ?mm toroid channels embedded within EXPECT guided cellular organization of cells seeded in these channels. In chondrogenic medium, actuated single mesenchymal stromal cells (MSCs) showed ~50 ?% narrower patterns by day 7, sustained to day 36 (p ?< ?0.001 vs. static, which widened to 137 ?± ?20 ?%). Actuated MSC spheroids elongated, forming bipedal shapes and fusing into extended patterns (length 480 ?± ?158 ?µm, p ?< ?0.0001) over 36 days. In 14-day human umbilical vein endothelial cells (HUVEC)-MSC co-cultures (10:1), actuation reduced pattern width by 27.5 ?% (p ?= ?0.0236), promoted early protrusions, and decreased cell circularity (vs. 2 ?% increase in static, p ?= ?0.0173), indicating enhanced elongation and potential vascularization. EXPECT's dynamic, actuation-mediated control of anisotropic cell organization overcomes limitations of static hydrogels, offering significant potential for engineering complex, organized tissues in regenerative medicine.