Synthetically Tractable Click Hydrogels for Three-Dimensional Cell Culture Formed Using Tetrazine–Norbornene Chemistry

Abstract

The implementation of bio-orthogonal click chemistries is a topic of growing importance in the field of biomaterials, as it is enabling the development of increasingly complex hydrogel materials capable of providing dynamic, cell-instructive microenvironments. Here, we introduce the tetrazine–norbornene inverse electron demand Diels–Alder reaction as a new cross-linking chemistry for the formation of cell laden hydrogels. The fast reaction rate and irreversible nature of this click reaction allowed for hydrogel formation within minutes when a multifunctional PEG-tetrazine macromer was reacted with a dinorbornene peptide. In addition, the cytocompatibility of the polymerization led to high postencapsulation viability of human mesenchymal stem cells, and the specificity of the tetrazine–norbornene reaction was exploited for sequential modification of the network via thiol–ene photochemistry. These advantages, combined with the synthetic accessibility of the tetrazine molecule compared to other bio-orthogonal click reagents, make this cross-linking chemistry an interesting and powerful new tool for the development of cell-instructive hydrogels for tissue engineering applications.

Versatile click alginate hydrogels crosslinked via tetrazine–norbornene chemistry

“…While recent work has used similar click chemistry for localized drug delivery, this work presents the first use of the tetrazine-norbornene click reaction to covalently crosslink polysaccharides into hydrogels [29,38]. Crosslinking of alginate by different methods has been extensively explored to make covalently crosslinked hydrogels that are mechanically robust, but these chemistries lack the cytocompatibility inherent in the bioorthogonal click reaction reported here [19,21,39].…”

“…In tissue engineering applications where cell trafficking within the hydrogel is desirable, click alginate hydrogels could be processed using existing techniques to introduce microscale porosity to the hydrogels [50,51]. Alternatively, click alginate polymers could be crosslinked using tetrazine or norbornene-modified matrix metalloproteinase-degradable peptide sequences to allow cell-mediated degradation [29,52]. The use of partially oxidized alginate polymers would also allow degradation of the hydrogel over controlled time scales for in vivo tissue engineering applications [20,53].The tissue compatibility and stability of click alginate hydrogels could make it particularly useful for applications where isolation from host immune cell infiltration is required [54,55].…”

“…These copper-free chemistries include strain-promoted azide-alkyne cycloaddition (SPAAC) and the inverse electron demand Diels-Alder reaction between tetrazine and norbornene [24,25]. Previous reports have used these click reactions primarily to crosslink click endfunctionalized branched polyethylene glycol (PEG) with linear crosslinkers composed of either PEG or linear peptides terminated with the appropriate click reaction pair [26][27][28][29]. The mechanical properties and swelling behavior of these click crosslinked PEG hydrogels could be tuned by varying the linear crosslinker concentration [30,31].…”

A microfluidic-based cell encapsulation platform to achieve high long-term cell viability in photopolymerized PEGNB hydrogel microspheres

“…PEGDA pre-polymer solutions were mixed to a final concentration of 10 wt% PEGDA ( M n ≈3400 Da, JenKem Technology, USA), 0.1 wt% LAP, and 5mM RGDS. The average molecular weight of each PEG chain was approximately conserved between PEGNB and PEGDA, and these materials have been shown to have similar mechanical properties 52,56-58 as hydrogels with comparative compositions. To characterize hydrogel microsphere size, a fluorescent dye, 0.01 wt% thiolated Rhodamine B, was added to and copolymerized with the hydrogel-forming solution to directly label the network.…”

Role of Surfactant during Microemulsion Photopolymerization for the Creation of Three-Dimensional Liquid Crystal Elastomer Microsphere Spatial Cell Scaffolds

“…inTrODUcTiOn Numerous biological and biocompatible synthetic materials have been developed for specific applications in tissue regeneration, and most are based on proteins, polysaccharides, polymers (e.g., PEG), peptides, or ceramic scaffolds (Koegler and Griffith, 2004;Kotov et al, 2004;Liu et al, 2005;Shanbhag et al, 2005;DeForest and Anseth, 2012;McCall et al, 2012;Alge et al, 2013;Lewis and Anseth, 2013;McKinnon et al, 2013). Any material that is designed for use in tissue regeneration, or cell studies in general, should promote adhesion, proliferation, and an environment for cell maturation.…”