Tuning the Morphological Properties of Granular Hydrogels to Control Lymphatic Capillary Formation

Introduction: : Lymphatic vessels play key roles in tissue homeostasis, immune regulation, and fluid balance. However, bioengineering strategies for regenerating lymphatic tissues have remained limited due to an incomplete understanding of how the extracellular matrix (ECM) regulates early lymphangiogenesis. Granular hydrogels, composed of jammed microgels, offer a tunable platform for creating 3D in vitro tissue models, yet their role in supporting lymphatic capillary morphogenesis remains unexplored. Norbornene-modified hyaluronic acid (NorHA) is a promising ECM-mimetic due to its biocompatibility, modularity, and interaction with LYVE-1 receptors on lymphatic endothelial cells (LECs). In this study, we investigate how microgel morphology and packing density in NorHA-based granular hydrogels affect lymphatic sprouting, vessel maturation, and network connectivity. We hypothesize that topological features such as curvature and pore size distribution will serve as instructive biophysical cues, guiding capillary self-assembly. These insights not only enhance our understanding of lymphatic development, but also inform the design of biomaterials optimized for lymphatic regeneration and integration in engineered tissues.

Materials and

Methods: : Granular hydrogels were fabricated using NorHA via two methods: vortexing and pipetting, the latter designed to produce uniform droplets using controlled flow and elliptical nozzles. Microgels were crosslinked with DTT via thiol-ene reactions, then centrifuged at varying speeds to generate loose (1500 g) and tight packing (6000 g). Resulting gels were characterized for porosity, pore size distribution, and mechanical properties using imaging and rheology. LECs were seeded within a degradable interstitial matrix infused into the granular gel, composed of RGD and MMP-cleavable crosslinkers. Growth factors (VEGF-C, FGF) were included to stimulate sprouting. After five days, gels were fixed and stained for VE-Cadherin, F-actin, and DAPI. Capillary structures were analyzed via confocal microscopy and 3D image reconstruction. Quantitative assessments of tube diameter, branching points, network length, and skeleton size were performed using AutoTube and ImageJ. Additionally, connectivity metrics such as node-to-node distance, capillary curvature, and cluster size were extracted from skeletonized images. Gene expression level was assessed via RT-qPCR to examine lymphatic maturation and matrix remodeling.

Results, Conclusions, and Discussions:: Granular gel morphology varied with the fabrication method. Pipetting microgels showed uniformity while vortexing exhibited wider size distributions (Fig. 1B). Gels’ porosity was reduced at higher centrifugation velocities (Fig. 1C). However, negligible effects were observed in pore size and pores density (Fig. 1D, E). Granular gels morphology significantly affected lymphatic tube formation. No capillaries formed under loose packing, while tight packing enabled capillary formation across all gel types (Fig. 2A). We observed similarities between granular gels and bulk NorHA hydrogels for capillaries features such as number of branches, tube width, and skeleton length (Fig. 2C-E). Nonetheless, the effective area occupied by vessels was increased for all the granular gels (Fig. 2B). Cluster size was significantly higher in pipetted gels, indicating enhanced capillary interconnectivity (Fig. 2H), correlating with their lower mean curvature and uniform topology (Fig. 2F). Capillary curvature mirrored that of gel droplets, especially in pipetted gels, suggesting structural guidance by ECM geometry. The analysis revealed a power-law decay in node-to-node distances (~17 µm), consistent with F-actin persistence length, indicating cytoskeletal alignment with gel features (Fig. 2G). RT-qPCR and ELISA confirmed early upregulation of MMP2, MMP14, and TIMP-1 for vortexing (Fig. 3A, B), whereas pipetting had gels showed later maturation. The secretion of reeling, an important lymphangiocrine, further indicated functional lymphatic constructs for granular gels (Fig. 3B). These findings demonstrate that granular hydrogel topology, particularly curvature and size distribution, regulates lymphatic capillary formation by guiding cellular organization and network complexity. In vivo studies showed integration of pre-grown lymphatic capillaries in pipetting granular gels with host's vasculature in murine models. This study underscores the importance of engineering microgel morphology in biomaterials design for tissue-specific vascularization, offering new strategies for lymphatic regeneration.

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