Norbornene Homopolymerization Limits Cell Spreading in Thiol–Ene Photoclick Hydrogels

Thiol-ene click chemistry is a powerful tool for engineering tissue-mimicking hydrogels permissive to 3D cell spreading. Thiol-norbornene chemistry allows precise control over crosslinking while seemingly avoiding alkene homopolymerization that can restrict 3D cell spreading. However, limited stress relaxation of a guest-host crosslinked norbornene-modified hyaluronic acid (NorHA) hydrogel employing a thiol-norbornene photoclick reaction prompts investigation into unintended norbornene homopolymerization. Norbornene conversion exceeds 1:1 thiol-ene expectations across various formulations, implicating homopolymerization. Reducing the number of norbornenes per NorHA chain (f) mitigates network formation via norbornene homopolymerization. Guest-host hydrogels fabricated with Nor₈HA (f = 8) exhibit 93.0 ± 1.6% relaxation, while those fabricated with Nor₄₀HA (f = 40) achieve only 42.3 ± 0.1% relaxation. As early as day 3 of culture, Nor₈HA hydrogels facilitate spreading of encapsulated human mesenchymal stromal cells (hMSCs) into a spindle-like morphology (aspect ratio: 2.95 ± 0.38), while Nor₄₀HA hydrogels appear to constrain cells into a spherical or compact star morphology (aspect ratio: 1.22 ± 0.01). Inference of a single-cell morphological space validates the two distinct hMSC morphological phenotypes primarily associated with polymer f. These results demonstrate that thiol-norbornene crosslinking is not fully stoichiometric in dilute aqueous systems and that network topology, modulated by f, is critical for restoring hydrogel permissivity and enabling cell spreading.