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 Materials (Impact Factor: 36.5). 07/2009; 8(8):659-64. DOI: 10.1038/nmat2473
Source: PubMed


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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    • "Our lab has already utilized click reactions for both microsphere formation and inter-microsphere cross-linking for scaffold stability [44]. Because copper, a common catalyst for these reactions, can be toxic to cells, we have focused on copper-free strain-promoted azide– alkyne cycloadditions, which have high conversions, fast kinetics, insensitivity to oxygen and water, stereospecificity, regiospecificity, and mild reaction conditions [45] [46] [47] [48]. "
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    ABSTRACT: Peripheral nerve regeneration is a complex problem that, despite many advancements and innovations, still has sub-optimal outcomes. Compared to biologically derived acellular nerve grafts and autografts, completely synthetic nerve guidance conduits (NGC), which allow for precise engineering of their properties, are promising but still far from optimal. We have developed an almost entirely synthetic NGC that allows control of soluble growth factor delivery kinetics, cell-initiated degradability and cell attachment. We have focused on the spatial patterning of glial-cell derived human neurotrophic factor (GDNF), which promotes motor axon extension. The base scaffolds consisted of heparin-containing poly(ethylene glycol) (PEG) microspheres. The modular microsphere format greatly simplifies the formation of concentration gradients of reversibly bound GDNF. To facilitate axon extension, we engineered the microspheres with tunable plasmin degradability. 'Click' cross-linking chemistries were also added to allow scaffold formation without risk of covalently coupling the growth factor to the scaffold. Cell adhesion was promoted by covalently bound laminin. GDNF that was released from these microspheres was confirmed to retain its activity. Graded scaffolds were formed inside silicone conduits using 3D-printed holders. The fully formed NGC's contained plasmin-degradable PEG/heparin scaffolds that developed linear gradients in reversibly bound GDNF. The NGC's were implanted into rats with severed sciatic nerves to confirm in vivo degradability and lack of a major foreign body response. The NGC's also promoted robust axonal regeneration into the conduit.
    Full-text · Article · Sep 2015 · Biomaterials
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    • "Various microfabrication 3D patterned cell-laden building blocks with natural and/or synthetic polymer have been widely adopted, such as photolithography [9] [10] [11], micromolding [12] and bioprinting [13] [14]. So far, the versatile and efficient cell-friendly photolithography allows fabrication of patterned building blocks with advantages of high precision, short time and low costs especially in fabrication of 3D patterned building blocks [15] [16] [17]. Various cells were seeded on patterned azido-chitosan hydrogel fabricated by UV lithography, such as cell spheroid microarrays of Hep G2 and NIH/3T3 [18], patterned cardiac fibroblast , cardiomyocyte and osteoblast microarrays on chitosan surfaces [19]. "
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    ABSTRACT: Natural and biodegradable chitosan with unique amino groups has found widespread applications in tissue engineering and drug delivery. However, its applications have been limited by the poor solubility of native chitosan in neutral pH solution, which subsequently fails to achieve cell-laden hydrogel at physiological pH. To address this, we incorporated UV crosslinking ability in chitosan, allowing fabrication of patterned cell-laden and rapid transdermal curing hydrogel in vivo. The hydrosoluble, UV crosslinkable and injectable N-methacryloyl chitosan (N-MAC) were synthesized via single-step chemoselective N-acylation reaction, which simultaneously endowed chitosan with well solubility in neutral pH solution, UV crosslinkable ability and injectability. The solubility of N-MAC in neutral pH solution increased 2.21-fold with substitution degree increasing from 10.9 % to 28.4 %. The N-MAC allowed fabrication of cell-laden microgels with on-demand patterns via photolithography, and the cell viability in N-MAC hydrogel maintained 96.3 ± 1.3%. N-MAC allowed rapid transdermal curing hydrogel in vivo within 60s through minimally invasive clinical surgery. Histological analysis revealed that low-dose UV irradiation hardly induced skin injury and acute inflammatory response disappeared after 7 days. N-MAC would allow rapid, robust and cost-effective fabrication of patterned cell-laden polysaccharide microgels with unique amino groups serving as building blocks for tissue engineering and rapid transdermal curing hydrogel in vivo for localized and sustained protein delivery. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Apr 2015 · Acta biomaterialia
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    • "The addition of the formed thiyl radicals to both electron rich and electron poor C@C double bonds displays the unique features of click reactions among them high yields, ambient process conditions, high reaction rates and insensitivity to water and oxygen [25]. Due to its versatility, highly efficient thiol–ene click chemistry has become implemented in numerous technologies that range from photopolymerization and photolithography to polymer functionalization strategies [26] [27] [28]. In addition, radical-mediated thiol– ene reactions together with catalyzed thiol-Michael reactions are widely used in ''grafting to'' and ''grafting from'' surface modifications of different types of polymer materials [29–31]. "
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    ABSTRACT: The present work highlights the use of UV induced thiol–ene click chemistry for designing elastomer surfaces with tailored friction properties. A two-step surface modification strategy based on two consecutive photoinduced thiol–ene reactions has been developed which enables controlled and patterned immobilization of micro-scaled inorganic particles onto diene-rubber surfaces. The influence of the coupled particles on both surface topography and surface roughness is determined by microscopic techniques whilst tribological studies are carried out to characterize the friction properties. The results give evidence that the attachment of selected micro-scaled particles provides a distinctive increase in surface roughness and a considerable decrease in the coefficient of friction. Both are influenced by the amount of particles immobilized onto the elastomer surface. The application of photolithographic techniques further provides elastomer materials with precisely and spatially controlled tribological properties. Whilst elastomer surfaces with randomly attached particles exhibit isotropic coefficients of friction, the tribological properties of micro-patterned elastomers are anisotropic.
    Full-text · Article · Feb 2015 · European Polymer Journal
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