Abstract 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 Nor 8 HA ( f = 8) exhibit 93.0 ± 1.6% relaxation, while those fabricated with Nor 40 HA ( f = 40) achieve only 42.3 ± 0.1% relaxation. As early as day 3 of culture, Nor 8 HA hydrogels facilitate spreading of encapsulated human mesenchymal stromal cells (hMSCs) into a spindle‐like morphology (aspect ratio: 2.95 ± 0.38), while Nor 40 HA 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.
Gentry et al. (Fri,) studied this question.