3D cell-laden hydrogels are increasingly used for in vitro and ex vivo cell expansion, controlled stem cell differentiation, and regenerative medicine. [1] Hydrogels can be fabricated by derivatives of purely synthetic polymers (e.g., poly(ethylene glycol) or PEG) and/or naturally derived biomacromolecules (e.g., gelatin, hyaluronic acid, or heparin). [2] Tumor basement membrane derived Matrigel is routinely used as a convenient matrix for 3D cell culture owing to its inherent cell affinity provided by laminin, collagen IV, and residual growth factors. However, Matrigel contains ill-defined and batch-to batch variability of extracellular matrix (ECM) components that may present challenges for studying the contributions of individual matrix cues on cell fate processes. To this end, synthetic PEG-based hydrogels afford a blank slate in which a bottom-up approach can be used to impart precise biochemical and biophysical properties in the cell-laden network. Advances in material chemistry have also enabled the conjugation of a variety of ECM motifs to the otherwise inert PEG-based network, as well as for spatiotemporal manipulation of matrix properties to facilitate the study of cell fate processes within a highly defined and controllable microenvironment. Chemically defined PEG-based hydrogels are particularly advantageous for studying iPSC behaviors in 3D. [3] For example, PEG hydrogels formed by Michael-type additions have proven effective in cell encapsulation owing to the reaction specificity between vinyl sulfone/maleimide (VS/MAL) and thiol (SH) moieties. Hubbell and colleagues utilized VS-SH reactions to determine the role of integrin activation on embryonic stem cell (ESC) self-renewal. [4] Lim et al. later use PEG-based VS-SH hydrogels for maintaining stemness of three human ESC lines. [5] Nonetheless, the reaction rates of Michael-type additions may be difficult to control and vary greatly from seconds to hours, depending on the type of Michael donor-acceptor pairs (e.g., VS-SH ≈ 30-120 min and MAL-SH ≈tens of seconds). [4a] Alternatively, enzymatic crosslinking provides excellent cytocompatibility and tunable gelation kinetics for in situ cell encapsulation. For instance, Xeno-free, chemically defined poly(ethylene glycol) (PEG)-based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene-click chemistries are integrated to form synthetic, dynamically tunable PEG-peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol-norbornene hydrogels crosslinked by multiarm PEG-norbornene (PEG-NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine-norbornene (Tz-NB) click reaction is then employed to dynamically stiffen the cell-laden hydrogels. Fast ...
Hydrogels cross-linked by inverse electron demand Diels–Alder (iEDDA) click chemistry are increasingly used in biomedical applications. With a few exceptions in naturally derived and chemically modified macromers, iEDDA click hydrogels exhibit long-term hydrolytic stability, and no synthetic iEDDA click hydrogels can undergo accelerated and tunable hydrolytic degradation. We have previously reported a novel method for synthesizing norbornene (NB)-functionalized multiarm poly(ethylene glycol) (PEG), where carbic anhydride (CA) was used to replace 5-norbornene-2-carboxylic acid. The new PEGNBCA-based thiol-norbornene hydrogels exhibited unexpected fast yet highly tunable hydrolytic degradation. In this contribution, we leveraged the new PEGNBCA macromer for forming iEDDA click hydrogels with [methyl]tetrazine ([m]Tz)-modified macromers, leading to the first group of synthetic iEDDA click hydrogels with highly tunable hydrolytic degradation kinetics. We further exploited Tz and mTz dual conjugation to achieve tunable hydrolytic degradation with an in vitro degradation time ranging from 2 weeks to 3 months. Finally, we demonstrated the excellent in vitro cytocompatibility and in vivo biocompatibility of the new injectable PEGNBCA-based iEDDA click cross-linked hydrogels.
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