4D force patterning enables spatial control of angiogenesis

Introduction: : Controlling vascular patterning is a fundamental challenge in developing tissue-engineered grafts for regenerative medicine, as defined vascular architecture is required for effective perfusion, integration, and function. Blood vessels align differently depending on the tissue context, such as (e.g., parallel in muscles or radially in retinas). Spatiotemporal variations in mechanical forces in vivo are believed to influence vascular alignment, morphology, and function. Mechanical strain is commonly studied in 2D monolayer models, where cells are stretched in 1- or 2- dimensions. However, these 2D models fail to capture the 3D changes in vascular architecture. A key barrier to understanding and directing vascular patterning is the difficulty of delivering tunable, non-invasive mechanical stimulation within dense 3D tissues. In particular, patterning forces dynamically in both 3D space and time (4D) to mimic evolving in vivo mechanical environments has been challenging. In vivo, endothelial cells experience mechanical stimuli in varying directions, intensities, and temporal dynamics based on their spatial arrangement.
The goal of this study is to leverage a non-invasive stimulation platform to drive the formation of new blood vessels with spatiotemporal control. Magneto-responsive hydrogels have emerged as a promising strategy to deliver mechanical stimulation without direct contact, as biological tissues are inherently insensitive to magnetic fields. We developed magnetic matrix actuation (MagMA), a platform that uses embedded magnetic microactuators within extracellular matrix-mimicking hydrogels to deliver tunable mechanical strain via an external permanent magnet. This system enables 4D spatiotemporal force patterning, offering a powerful strategy to control angiogenesis within engineered tissues.

Materials and

Methods: : Our MagMA-on-Chip platform features a hollow channel lined with human umbilical vein endothelial cells (HUVECs), embedded within a collagen matrix containing a magnetic microactuator (Fig. 1A). To achieve programmable mechanical stimulation, we integrated a three-axis actuator to move magnets above the devices with precise control over height, speed, and direction (Fig. 1B). This setup enabled the tunable delivery of strain magnitude, frequency, and direction across distinct vessel regions, mimicking the heterogeneous mechanical environments observed in vivo.
Using our three-axis actuator, we systematically varied mechanical strain parameters–magnitude (5% vs 15%), frequency (static vs 1Hz), onset timing (day 1 vs day 3), and direction–to identify the combinations that enhance angiogenic sprouting and vascular barrier function over a 7-day culture. Permeability was assessed by perfusing devices with 10kDa fluorescent dextran, imaging real-time diffusion into the ECM, and quantifying using MATLAB. Sprouts were quantified by fixing and staining for PECAM-1 and F-actin, followed by skeletonization in ImageJ and network analysis (sprout length, number of sprouts, and branches) using Mathematica.
To characterize 3D spatial differences in sprouting, confocal z-stacks were reconstructed in IMARIS, and a filament tracing algorithm segmented individual sprouts from the parent vessel. Sprout position and length were mapped to assess regional angiogenic differences across vessel quadrants (e.g., magnet, non-magnet, top, and bottom regions). To identify molecular pathways responsive to mechanical strain, we are currently performing RNAseq on samples from two strain conditions and an unstimulated control, aiming to uncover differentially expressed genes that link mechanical cues to vascular structure and function.

Results, Conclusions, and Discussions:: Our 4D vascular engineering platform enabled us to investigate how strain parameters (e.g., magnitude, frequency, direction) regulate vascular architecture and function over time. We investigated 0% (no), 5% (low), and 15% (high) strain (Fig. 1C). Low-strain conditions significantly increased sprout formation by 5-fold compared to the no-strain control. In contrast, high-strain conditions resulted in a 2-fold reduction in sprout number; however, these sprouts exhibited increased length (Fig. 1D). Additionally, endothelial cell–cell junctions showed notable morphological and functional alterations under varying mechanical strain conditions (Fig. 1E). Specifically, under high strain, junctions became less circular compared to controls, accompanied by a functional decrease in vessel permeability by 55% (Fig. 1F). We are currently leveraging RNAseq to identify differentially expressed genes in low, high, and no strain.
In addition to programming the magnitude of angiogenesis, we observed that dynamic mechanical stimulation also regulates the direction of growth. Spatial analysis using 3D reconstruction demonstrated that x-axis strain preferentially promoted sprouting along the magnet–non-magnet axis, compared to the top-bottom regions (Fig. 1G). Leveraging our 4D vascular engineering platform, we dynamically orchestrated multi-directional sprouting by altering the direction of mechanical stimulation. Specifically, switching the stimulation axis perpendicular to the initial orientation resulted in realignment of emerging sprouts along both directions, giving rise to distinct ‘L-shaped’ vascular structures (Fig. 1H).
Through 4D dynamic and tunable mechanical stimulation, this platform holds potential for engineering vasculature with programmable architecture and functionality—critical for matching the distinct structural and functional demands of different organs in tissue engineering.

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