M.Sc. Therese Steudter

Hydrogels mimicking the mechanical and biochemical features of the cellular microenvironment allow cell encapsulation and facilitate in vitro 3D culture. In addition to biocompatibility and reactivity in physiological conditions, a key criterion for crosslinking chemistry is appropriate gelation kinetics to allow mixing and homogeneous distribution of cells with the hydrogel precursors. We have previously presented aryl methylsulfone/thiol (MS/SH) reaction as a thiol-reactive cross-linking system for cell encapsulation in star polyethylene glycol (PEG4) hydrogels with a gelation kinetics in minutes time scale. Remaining experimental challenges for this system are a finer modulation of gelation kinetics and streamlining the synthesis of the prepolymer. Here we present the possibility to tune the gelation kinetics by introducing an electron-withdrawing substituent at p-position of the aryl MS ring. This variant also presents synthetic advantages. We study the influence of the p-substituent on the physicochemical properties of MS/SH crosslinked hydrogels, and their performance for cell encapsulation. We compare these properties with the PEG-MS variant containing an electron-donating linker. The new star poly(ethylene glycol)-4-(5-(methylsulfonyl)-1H-tetrazol-1-yl)benzamide (PEG4-CONH-TzMS) shows superior properties as cell encapsulating hydrogel in terms of ease of mixing polymer precursors, faster gelation, homogenous cell distribution and high enzymatic stability.

Excessive protease activity and impaired tissue regeneration are hallmarks of many disease states. Elevated matrix metalloproteinase-9 (MMP-9) plays a key role in adverse tissue remodeling by excessively degrading extracellular matrix (ECM) components and growth factors. Tissue inhibitor of metalloproteinase-3 (TIMP-3) regulates ECM turnover, and its bioavailability is influenced by glycosaminoglycans (GAGs). This study aimed to develop a methacrylated gelatin (GelMA)-based hydrogel functionalized with acrylated sulfated hyaluronan (sHAc) as a TIMP-3 delivery system to decrease ECM degradation under pathophysiological conditions. sHAc incorporation enhanced hydrogel stiffness, reduced degradation rates and yielded sustained TIMP-3 release for up to 28 days. Molecular modeling and surface plasmon resonance demonstrated preferential binding of TIMP-3 to sHAc over hyaluronan methacrylates, together providing a molecular rationale for the reduced and sustained release of TIMP-3 from sHAc-containing hydrogels. Angiogenesis-related functional assays, supported by molecular modeling studies, indicate that sHAc modulates the anti-angiogenic activity of TIMP-3 by altering vascular endothelial growth factor receptor-associated signaling, while preserving metalloproteinase inhibition. Released TIMP-3 from GelMA/sHAc hydrogels retained bioactivity, effectively inhibiting MMP-9 activity and mitigating ECM degradation in-vitro and in human ex-vivo models. In a murine subcutaneous implantation model, sHAc-functionalized TIMP-3-loaded hydrogels were associated with reduced inflammatory cell presence and altered vascular- and matrix-related tissue signatures compared with GelMA controls. These findings underscore the potential of sHAc-functionalized GelMA hydrogels as biomaterials for therapeutics delivery, offering controlled TIMP-3 release and sustained bioactivity to promote ECM stability and on-demand MMP inhibition. This system represents a promising strategy for addressing the challenges of excessive MMP activity.

Photocrosslinkable formulations based on the radical thiol-ene reaction are considered better alternatives than methacrylated counterparts for light-based fabrication processes. This study quantifies differences between thiol-ene and methacrylated crosslinked hydrogels in terms of precursors stability, the control of the crosslinking process, and the resolution of printed features particularized for hyaluronic acid (HA) inks at concentrations relevant for bioprinting. First, the synthesis of HA functionalized with norbornene, allyl ether, or methacrylate groups with the same molecular weight and comparable degrees of functionalization is presented. The thiol-ene hydrogel precursors show storage stability over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrate up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels crosslinked with a dithiol allowed printing of 13.5 × 4 × 1 mm3 layers with holes of 100 µm resolution within 2 s. This is the smallest feature size demonstrated in DLP printing with HA-based thiol-ene hydrogels. The results are important to estimate the extent to which the synthetic effort of introducing –ene functions can pay off in the printing step.