Poster BB7 - Engineering Granular Hydrogels for Lymphatic Vasculature Formation: Effect of Gels Morphology on the Development of Perfusable Vasculature Constructs

Introduction: Biomimetic hydrogels have emerged as promising candidates due to their ability to mimic the extracellular matrix and create microenvironments that can support tissue morphogenesis, and specifically vasculature formation which is crucial during wound healing. In particular, engineering lymphatic vasculature holds significant promise for applications in lymphatic regeneration and disease modeling. However, conventional bulk hydrogels have limitations on supporting cell invasion while providing mechanical stability necessary for vessels maintenance. Conversely, granular hydrogels made of microgels subunits have a great potential in promoting lymphangiogenesis as they can create conducive microenvironments with mechanical support without the inclusion of mesodermal cells. However, understanding the influence of granular gel morphology on lymphatic sprout formation and functionality is crucial for optimizing their performance in tissue engineering applications.

Materials and

Methods: We investigated the morphological effects of granular gels on the development of perfusable lymphatic vasculature constructs. Using norbornene-modified hyaluronic acid (NorHA) polymer and DTT as crosslinker, microgels were fabricated via pipetting and vortexing techniques yielding distinct morphologies. The different morphological features of the granular gels were characterized through confocal microscopy and image analysis. The effect of the gels’ morphology on the rheological properties of the materials was assessed. A degradable interstitial matrix made with NorHA and integrating an MMP-sensitive crosslinker and RGD was incorporated with lymphatic endothelial cells (LECs) in suspension at 6x106, 8x106 and 10x106 cells/mL. Lymphatic sprouting was assessed via immunostaining and image analysis for capillaries quantification.

Results, Conclusions, and Discussions: Visually, the granular gels produced by different methods have different particle and interstice features (Fig. 1A, B). Morphological analysis revealed that vortexing-produced gels exhibited higher porosity (Fig. C, II), but wider microgel size distribution compared to pipetting (Fig. C, I), resulting in smaller pores (Fig. C, III) and tighter packing (Fig. C, IV). Rheological analysis showed that vortexing gels had a higher storage modulus, indicating enhanced mechanical properties due to increased interparticle contact. Assessing the impact of the morphological variances of the granular hydrogels on lymphatic sprout formation, we observed significant differences in sprout morphology between pipetting and vortexing gels (Fig. 1D). Pipetting gels had lower number of branches (Fig. E, II) but a higher area covered with vessels (Fig. C, I) related to a greater tube width (Fig. C, III). Gene expression analysis further supported these observations, with pipetting gels showing upregulation of lymphatic markers, being 5-fold the reference level for lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and 3-fold for prospero homeobox 1 (Prox1) during early-stage lymphatic sprout formation. Finally, perfusion through the vasculature constructs was tested via needle injection of fluorescent beads. The tests showed the formation of functional lymphatic vessels with lumen-like structure.

Overall, our findings provide novel insights into factors influencing lymphangiogenesis within granular hydrogels, demonstrating the potential to generate lymphatic tube formation without the need for supporting cells. By elucidating the morphological effects on lymphatic vasculature development and functionality, our study contributes to the development of platforms for regenerative medicine and modeling of disease progression. These insights pave the way for the design of biomimetic materials capable of promoting lymphatic regeneration and advancing therapeutic strategies for lymphatic-related pathologies.

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