Introduction: Synthetic biomaterials typically present an elastic, mechanically static environment to cells, despite tissues being viscoelastic and spatiotemporally heterogeneous. Only a few approaches have explored such complexity[1],[2]. Here, we developed hyaluronic acid (HA) hydrogels with independent control of viscoelasticity over time and space to investigate human mesenchymal stem cell (MSC) spreading, actin organization, and Yes-associated protein (YAP) signaling.
Materials and Methods: HA was modified with norbornene (Nor), β-cyclodextrin (CD), and/or adamantane (Ad) groups. Di-thiol crosslinkers, thiolated Ad, and/or thiolated RGD (2 mM) were coupled to norbornene groups via UV light-mediated thiol-ene addition. NorHA was photocrosslinked to create elastic, covalently crosslinked gels (Fig 1A). CDHA was mixed with AdNorHA and then photocrosslinked to create viscoelastic gels containing dynamic, supramolecular Ad and CD interactions[3], as well as covalent crosslinks between the AdNorHA chains only (Fig 1A). Gel mechanics were measured by rheometry and AFM. MSC (Lonza) spread area, cell shape index (CSI), YAP nuclear/cytoplasmic intensity ratio, and F-actin organization were quantified in NIH ImageJ for at least 50 cells per experimental group (*** denotes p < 0.001). Light-mediated spatiotemporal manipulation of viscoelasticity was achieved by either introducing di-thiol crosslinkers to increase covalent crosslinking between Nor groups or thiolated Ad peptide where the thiol reacts to Nor functionalities and the Ad interacts with CD groups to increase supramolecular crosslinking without altering covalent crosslinking.
Results and Discussion: Elastic and viscoelastic hydrogels with variable crosslinking dynamics but equivalent HA content and elastic moduli were fabricated (E ~ 1, 5, and 15 kPa). Viscoelastic gels displayed stress relaxation of ~ 35% for an initial 10% strain, which was not observed in elastic gels (Fig 1B). Additionally, loss moduli (G”) were consistently an order of magnitude higher for viscoelastic over elastic gels with equivalent storage moduli (G’, Fig 1B). Gel degradability could also be controlled by using either protease-degradable or non-degradable di-thiol peptide crosslinkers. While MSCs displayed similar spread area and shape between elastic and viscoelastic gels at each respective crosslink density, F-actin stress fiber organization and YAP nuclear/cytoplasmic ratio were significantly higher (p < 0.001) for MSCs on the low (E ~ 1 kPa) and middle (E ~ 5 kPa, Fig 1C, scale bar = 50 μm) crosslinking density viscoelastic gels (1.41 ± 0.04; 1.88 ± 0.05) compared to elastic variants (1.16 ± 0.02; 1.49 ± 0.04). Spatiotemporal tuning of covalent and supramolecular crosslinking was achieved by either altering UV exposure during secondary photocrosslinking (covalent, Fig 1D) or by incorporating thiolated Ad peptide to increase Ad-CD interactions (supramolecular, Fig 1D).
Conclusion: We developed a hydrogel platform based on control over HA crosslinking dynamics to fabricate cell culture substrates with tunable mechanics (stiffness, viscoelasticity). MSCs cultured on substrates of low to intermediate stiffness (E ~ 1-5 kPa) displayed similar spread area and shape but significantly increased YAP nuclear localization on viscoelastic gels. The viscoelasticity could then be controlled over time and space to add further complexity to these in vitro cellular environments.
References:
[1] Cameron AR, Biomaterials, 2011, 32(26): 5979-93
[2] Chaudhuri O, Nat Commun, 2015, 6: 6365
[3] Rodell CB, Biomacromolecules, 2013, 14(11): 4125-34