Assistant Professor Tulane University Destrehan, Louisiana, United States
Introduction: : Stem cell therapy offers curative potential for hematologic and metabolic disorders such as sickle cell disease and diabetes; however, its clinical application remains limited by challenges in targeted delivery, including premature clearance and reduced cell viability. Granular hydrogels, due to their tunable mechanical properties and self-healing behavior, represent a promising strategy to overcome these barriers by protecting stem cells during infusion and enhancing their engraftment within their respective niches. Our work focuses on engineering hydrolytically degradable polyethylene glycol (PEG)-based granular hydrogels with tunable degradation profiles to enable effective encapsulation and localized delivery of stem cells for regenerative therapies. In parallel, we are developing an in vitro degradation model that recapitulates the physiological constraints and microenvironment of the bone marrow cavity, providing a predictive platform to optimize material performance prior to in vivo application.
Materials and
Methods: : Hydrolytically degradable 4-arm PEG macromers were synthesized via a two-step functionalization strategy. In the first step, a thiol-Michael addition was carried out between 4-arm PEG acrylate (with variable molecular weights) and a heterobifunctional thiol-amine, either PEG-thiol amine or cysteamine, in the presence of dimethylphenylphosphine (DMPP) as a nucleophilic catalyst(Fig1). This reaction produced PEG intermediates bearing β-thioester bonds and terminal primary amines. In the second step, these amine-terminated intermediates were reacted with 3-maleimidopropionic acid N-hydroxysuccinimide (NHS) ester to introduce terminal maleimide groups via NHS-mediated amidation. Macromer identity and degree of functionalization at each step were confirmed by proton NMR spectroscopy. The granular hydrogels were then formed with a 300um nozzle fluid focusing device consisting three independent flow channels of macromer solution, oil mixture for emulsification and crosslinker, meeting at junctions to promote formation of monodispersed crosslinked microgels1. Gel sizes were measured using light microscopy and quantified using imagej software.Gel fraction was measured post-crosslinking, and a microfluidic perfusion model (“micromodel”) was engineered with a center channel to hold microgels and media reservoir channels flanking on both sides to mimic conditions in bone marrow sinusoids and trabecular spaces. The device supports continuous perfusion at flow rates of 0.1–1 µL/min, which recapitulates the physiological range of interstitial fluid velocities observed in the bone marrow niche (typically 0.1–5.0 µm/s)2. This design enables direct assessment of hydrogel swelling and degradation kinetics under dynamic, tissue-relevant conditions, providing a predictive platform for stem cell scaffold behavior in vivo.
Results, Conclusions, and Discussions:: PEG-based microgels with variable spatial positioning of hydrolytically degradable sites were successfully created and confirmed by proton NMR, showing the disappearance of acrylate protons and appearance of maleimide proton peaks at respective stages(Fig.2). Microgel generated using a fluid focusing microfluidic device yielded uniform crosslinked droplets(Fig. 3). Quantitative analysis of microgel size distribution and polydispersity index (PDI) is currently in progress; preliminary image analysis indicates a monodisperse population with sizes in the expected microscale range (mean diameter: 458.83 µm; PDI: 0.49; n=247). Functional characterization is proceeding in a custom microfluidic perfusion platform that reproduces bone‑marrow‑level interstitial flow. In this setting, swelling ratio, gel fraction, and mass‑loss kinetics are being quantified to test the central hypothesis that terminally placed β‑thioesters and lower‑molecular‑weight precursors accelerate hydrolytic degradation relative to internally placed linkages and higher‑molecular‑weight formulations. Early qualitative observations confirm that network architecture is a dominant determinant of stability; full statistical analyses are in progress and will be reported.
Collectively, these data will establish a versatile, flow‑compatible method for producing structurally tunable PEG granular hydrogels. Completion of the pending measurements will delineate design rules linking macromer chemistry to degradation rate and swelling behaviour, information critical for matching scaffold disassembly to the temporal requirements of stem‑cell engraftment. Subsequent work will integrate hematopoietic stem cells to evaluate viability, retention, and functional activity under dynamic perfusion, positioning this platform for translational studies targeting disorders such as sickle‑cell disease.
Acknowledgements and/or References (Optional): : Acknowledgements Many thanks to members of the Tissue Engineering and Microphysiological Systems (TEMPs) laboratory, especially Ethan Bryne, PhD and Mark Mondrinos, PhD, for their resources and training in microfluidic device fabrication.
References (1) GB, T.; AE, G.; VM, L.; AC, N.; BA, P.; A, M.-B.; JF, S.; AJ, G.; BAC, H. Gelatin Maleimide Microgels for Hematopoietic Progenitor Cell Encapsulation - PubMed. J. Biomed. Mater. Res. A 2024, 112 (12). https://doi.org/10.1002/jbm.a.37765. (2) Human bone perivascular niche-on-a-chip for studying metastatic colonization. https://doi.org/10.1073/pnas.1714282115. Images]
