Sequential Click Reactions for Synthesizing and Patterning 3D Cell Microenvironments

Department of Chemical and Biological Engineering, University of Colorado, UCB Box 424 Boulder, Colorado 80309-0424, USA.
Nature Material (Impact Factor: 36.43). 07/2009; 8(8):659-64. DOI: 10.1038/nmat2473
Source: PubMed

ABSTRACT Click chemistry provides extremely selective and orthogonal reactions that proceed with high efficiency and under a variety of mild conditions, the most common example being the copper(I)-catalysed reaction of azides with alkynes. While the versatility of click reactions has been broadly exploited, a major limitation is the intrinsic toxicity of the synthetic schemes and the inability to translate these approaches into biological applications. This manuscript introduces a robust synthetic strategy where macromolecular precursors react through a copper-free click chemistry, allowing for the direct encapsulation of cells within click hydrogels for the first time. Subsequently, an orthogonal thiol-ene photocoupling chemistry is introduced that enables patterning of biological functionalities within the gel in real time and with micrometre-scale resolution. This material system enables us to tailor independently the biophysical and biochemical properties of the cell culture microenvironments in situ. This synthetic approach uniquely allows for the direct fabrication of biologically functionalized gels with ideal structures that can be photopatterned, and all in the presence of cells.

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    • "Recently, ''4-D'' hydrogels that enable spatial and temporal patterning of specific ligand geometries in 3-D—the ''fourth dimension'' being time—have been developed in order to guide cellular behaviours such as polarization and migration in real time [124]. For example, Anseth and colleagues exploited the orthogonality between the copper-free azide-alkyne and thiol–ene systems by creating a PEG hydrogel—formed via the former reaction—that could be selectively [125] and reversibly [126] photo-patterned using the latter reaction, post-gelation. In addition to these and other PEG-based hydrogels [127] [128] [129], photo-patterning has also been achieved in ligand-functionalized agarose [130] [131] [132] [133], hyaluronic acid [134] [135] and alginate [136] hydrogels. "
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    ABSTRACT: The role of soluble messengers in directing cellularbehaviours has been recognised for decades. However, many cellularprocesses including adhesion, migration and stem cell differentiation arealso governed by chemical and physical interactions with non-soluble components ofthe extracellular matrix (ECM). Amongst other effects, a cell's perception of nano-scale features such as substrate topography and ligand presentation, andits ability to deform the matrix via the generation of cytoskeletal tension play fundamentalroles in these cellular processes. As a result, many biomaterials-based tissue engineering and regenerativemedicine strategies aim to harness the cell's perception of substrate stiffness and nano-scale features to direct particular behaviours. However, since cell-ECM interactions vary considerablybetween two-dimensional (2D) and three-dimensional (3D) models, understanding their influence over normal and pathological cell responses in3D systems which better mimic thein vivomicroenvironment is essentialto efficiently translate such insights into medical therapies.This reviewsummarises the key findings in these areas and discusses how insights from 2D biomaterials are being utilised to examinecellular behaviours in more complex 3D hydrogel systems, in which not only matrix stiffness, but also degradability plays an important role, and in which defining the nano-scale ligand presentation presents an additional challenge.
    Acta Biomaterialia 10/2014; 11:3–16. DOI:10.1016/j.actbio.2014.09.038 · 5.68 Impact Factor
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    • "The biochemical cues could also be removed by exposure to UV light, giving excellent spatiotemporal control over the presentation of Fig. 1. Preparation and post-modification of cytocompatible SPAAC click hydrogels. (A) Preparation of 3D network hydrogels via SPAAC reaction from 4-arm PEG tetra-azide and difunctionalized peptide sequence; (B) effect of patterned RGD on 3T3 population within 3D click hydrogels that formed using full mask, no mask or full mask with a 250-mm-square opening (illustrated by the dashed lines); cells adopt a spread morphology only in user-defined regions of RGD [18]. biologically relevant chemical cues within the hydrogels. "
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    ABSTRACT: Hydrogels, microgels and nanogels have emerged as versatile and viable platforms for sustained protein release, targeted drug delivery, and tissue engineering due to excellent biocompatibility, a microporous structure with tunable porosity and pore size, and dimensions spanning from human organs, cells to viruses. In the past decade, remarkable advances in hydrogels, microgels and nanogels have been achieved with click chemistry. It is a most promising strategy to prepare gels with varying dimensions owing to its high reactivity, superb selectivity, and mild reaction conditions. In particular, the recent development of copper-free click chemistry such as strain-promoted azide-alkyne cycloaddition, radical mediated thiol-ene chemistry, Diels-Alder reaction, tetrazole-alkene photo-click chemistry, and oxime reaction renders it possible to form hydrogels, microgels and nanogels without the use of potentially toxic catalysts or immunogenic enzymes that are commonly required. Notably, unlike other chemical approaches, click chemistry owing to its unique bioorthogonal feature does not interfere with encapsulated bioactives such as living cells, proteins and drugs and furthermore allows versatile preparation of micropatterned biomimetic hydrogels, functional microgels and nanogels. In this review, recent exciting developments in click hydrogels, microgels and nanogels, as well as their biomedical applications such as controlled protein and drug release, tissue engineering, and regenerative medicine are presented and discussed.
    Biomaterials 03/2014; 35(18). DOI:10.1016/j.biomaterials.2014.03.001 · 8.31 Impact Factor
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    • "The recent expansion of the click chemistry library suggested orthogonal and mixed-mode reaction strategies, already utilized in the synthesis of hydrogels, which could provide a combinatorial library for the synthesis of BioSIN. The demands for the construction of such complex synthetic macromolecular structures have already led to double and triple click reactions [11] [13] [14]. The Cucatalyzed azide-alkyne cycloaddition is by far the most pervasive in hydrogel synthesis [15] [16]; nevertheless, the presence of toxic copper during the Cu-catalyzed azide-alkyne cycloaddition can be cytotoxic and limits its use in the synthesis of products for cell encapsulation. "
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    ABSTRACT: The integration of biological extracellular matrix (ECM) components and synthetic materials is a promising pathway to fabricate the next generation of hydrogel-based tissue scaffolds that more accurately emulate the microscale heterogeneity of natural ECM. We report the development of a bio/synthetic interpenetrating network (BioSINx), containing gelatin methacrylamide (GelMA) polymerized within a poly(ethylene glycol) (PEG) framework to form a mechanically robust network capable of supporting both internal cell encapsulation and surface cell adherence. The covalently crosslinked PEG network was formed by thiol-yne coupling, while the bioactive GelMA was integrated using a concurrent thiol-ene coupling reaction. The physical properties (i.e. swelling, modulus) of BioSINx were compared to both PEG networks with physically-incorporated gelatin (BioSINP) and homogenous hydrogels. BioSINx displayed superior physical properties and significantly lower gelatin dissolution. These benefits led to enhanced cytocompatibility for both cell adhesion and encapsulation; furthermore, the increased physical strength provided for the generation of a micro-engineered tissue scaffold. Endothelial cells showed extensive cytoplasmic spreading and the formation of cellular adhesion sites when cultured onto BioSINx; moreover, both encapsulated and adherent cells showed sustained viability and proliferation.
    Biomaterials 12/2013; 35(6). DOI:10.1016/j.biomaterials.2013.11.009 · 8.31 Impact Factor
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