We have developed a new type of biomimetic, user-modifiable, and cell-adaptable hydrogel matrices via the combination of Michael-type addition reaction and the rapid cycloaddition of s-tetrazines (Tz) with strained trans-cyclooctenes (TCO). A hydrophilic random copolymer carrying both acrylate and tetrazine groups (POM-AT) was mixed with a high molecular weight thiolated hyaluronic acid (HA-SH) to establish the initial Michael network compatible with in situ encapsulation of human mesenchymal stem cells (hMSCs). A matrix metalloprotease (MMP)-degradable bis-acrylamide crosslinker (GIW-bisAM) was included in the Michael network to enable hMSC-mediated remodeling of the synthetic environment. The cell-laden gel constructs were subsequently modified with TCO-conjugated HA (HA-TCO) or TCO-conjugated cell-adhesive peptide (RGD-TCO) via a diffusion controlled interfacial reaction mechanism on day 2 or day 6, and the constructs were maintained in hMSC growth media for a total of 9 days. hMSCs cultured in MMP-degradable Michael network remained rounded, but developed cellular processes, adopted elongated morphology. Analysis of the cellular constructs by real-time polymerase chain reaction revealed an upregulation of MMP1, and downregulation of markers for cell differentiation for RGD-TCO-modified constructs, as compared to controls without any modification. Stiffening the Michael network by juxtaposition of the <u>t</u>etrazine network using HA-TCO did not significantly alter cell morphology. Our approach allows facile manipulation of synthetic extracellular matrices in real time to promote cell growth and tissue morphogenesis.

Real-time, bioorthogonal tuning of cell-laden hydrogels to modulate stem cell behaviors in 3D

256th National Meeting (2018)

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