Degradable multi-arm PEG hydrogels with tunable stiffness and diffusivity.

The bone marrow extracellular matrix (BM-ECM) has distinct mechanical and biochemical subniches, in which hematopoietic stem cells (HSCs) reside, self-renew, and differentiate into immune cells. Stiffness and diffusivity are key mechano-regulators of HSC fate. To control these key microenvironment parameters, we prepared a poly(ethylene-glycol) norbornene (PEGNB)-modified hydrogel platform with tunable stiffness and solute diffusivity by varying the polymer volume fraction, the number of macromolecular arms, their arm length, and their crosslinker type. The latter was identified as either nondegradable dithiothreitol (DTT) or matrix metalloproteinase (MMP)-degradable peptides. We characterized 24 PEGNB hydrogels for stiffness, swelling, and solute diffusion. Hydrogel swelling ratios ranged from 0.89 to 2.48 for DTT-crosslinked networks and 1.49 to 4.86 for peptide-crosslinked networks, supporting solvent retention for cell culture. Storage moduli ranged from 6.4 to 44.8 kPa (DTT-crosslinked networks) and 3.6 to 32.7 kPa (peptide-crosslinked networks), within the physiological range of the BM-ECM. Solute diffusivity values were also evaluated for all hydrogel formulations. Introduction of an MMP-sensitive crosslinker maintained the same relationship among hydrogel stiffness, swelling and solute diffusion while allowing for cell-mediated remodeling and high cell viability. Unlike prior predictive models, our study accounts for structural complexity to better match in vivo conditions. Therefore, we present a modular framework for engineering PEGNB hydrogels with independently tunable mechanical and transport properties, providing a robust, physiologically relevant platform to investigate stem cell-matrix interactions and advance stem cell-based tissue engineering.