(275c) Engineering Granular Hydrogels for Lymphatic Vasculature Formation: Effect of Gels Morphology on the Development of Perfusable Vasculature Constructs

Advancements in tissue engineering and regenerative medicine permitted the development of biomimetic materials capable of fostering complex cellular interactions and tissue growth. 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.

In this study, 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. Morphological analysis revealed that vortexing-produced gels exhibited higher porosity but wider microgel size distribution compared to pipetting, resulting in tighter packing and smaller pores. Rheological analysis showed that vortexing gels had a higher storage modulus, indicating enhanced mechanical properties due to increased interparticle contact.

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. Interestingly, all the granular hydrogels groups were able to sustain the formed vessels for longer times than its bulk hydrogel counterparts. 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. Pipetting gels facilitated linear-like sprouts, while vortexing gels exhibited enhanced microparticle coverage. 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. Moreover, we explored the influence of interstitial matrix composition on sprout morphology and gene expression levels. Altering the composition of the interstitial matrix resulted in distinct sprout morphologies, highlighting the importance of synchronized morphological and compositional tuning in granular hydrogel design. 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 early-stage lymphatic sprout 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.