bioRxiv2025
Engineering organized microvascular networks remains a critical challenge in tissue engineering and regenerative medicine. While biochemical approaches for patterning angiogenesis via growth factor delivery have shown promise, their inability to pattern sustained growth factors with spatiotemporal control limits effectiveness. Here, we demonstrate that dynamically patterned mechanical forces enable precise spatiotemporal control over angiogenic sprouting. We developed a magnetically actuated human vessel-on-a-chip platform that integrates a perfusable endothelialized microchannel within a collagen matrix and allows non-invasive and tunable mechanical stimulation across three spatial dimensions and time (4D). Using an automated 3-axis actuator, we systematically investigated how strain magnitude, frequency, and direction modulate endothelial cell behavior and vessel morphogenesis. Dynamic mechanical stimulation at physiological strain magnitudes (5-15%) enhanced endothelial alignment and barrier function while promoting angiogenesis in a strain-magnitude-dependent manner: lower dynamic strain (5%) maximized sprout initiation, whereas higher dynamic strain (15%) promoted elongation of sprouts. Sequential reorientation of strain direction reprogrammed sprouting trajectories along X, Y, and Z directions, generating complex sprout geometries such as L-shaped branches. RNA sequencing revealed mechanically induced transcriptional profiles distinct from unstimulated controls, characterized by upregulation of genes associated with angiogenesis, mechanotransduction, and extracellular matrix remodeling. Functional perturbation of Piezo1 reduced strain-induced sprouting without altering barrier stabilization, indicating that dynamic mechanical stimulation engages multiple mechanotransduction pathways to regulate angiogenesis. Collectively, these findings establish a strategy for spatiotemporally controlled angiogenesis through 4D force patterning to program vascular morphogenesis while preserving function. This approach provides a foundation for engineering hierarchically organized vascular networks for tissue regeneration. SignificanceGeneration of spatially organized, perfusable microvascular networks is essential for building functional human tissues. Biochemical approaches to pattern angiogenesis rely on diffusive growth factors, which limit control over spatiotemporal sprouting dynamics. Here, we demonstrate that dynamically patterned mechanical forces direct vascular morphogenesis across three spatial dimensions and time (4D). Using a magnetically actuated human vessel-on-a-chip, we show how strain magnitude and orientation govern angiogenic sprouting and reveal transcriptional programs linking mechanical cues to observed functional changes. For the first time, we show that dynamic reorientation of imposed forces can reprogram angiogenic trajectories in real-time. This platform enables programmable mechanical control of angiogenesis and systematic dissection of mechanotransduction pathways, advancing strategies for tissue vascularization, and modeling mechanically regulated vascular diseases.