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Chemical reaction systems that can occur via multiple pathways in a controllable fashion are highly attractive for advanced materials applications and biological research. In this report, we introduce a bioorthogonal reaction manifold based on a chalcone pyrene ( CPyr ) moiety that can undergo either red-shifted photoreversible [2 + 2] cycloaddition or thiol-Michael addition click reaction. By coupling the CPyr to a water-soluble poly(ethylene glycol) end group, we demonstrate the efficient polymer dimerization and cleavage by blue light (λ = 450 nm) and UV light (λ = 340 nm), respectively. In the absence of light, CPyr rapidly reacts with thiols in aqueous environments, enabling fast and efficient polymer end-group functionalization. The chemical reaction manifold was further employed in polymer cross-linking for the preparation of hydrogels whose stiffness and morphology can be modulated by different photonic fields or the addition of a thiol cross-linker. The photoreversible cycloaddition and thiol-Michael addition click reaction can be used in conjunction for spatial and temporal conjugation of a streptavidin protein. Both cross-linking conditions are nontoxic to various cell lines, highlighting their potential in biomaterials applications.

Solvent-free photopolymerization of vinyl monomers to produce high modulus materials with applications in 3D printing and photoswitchable materials is demonstrated. Polymerizable eutectic (PE) mixtures are prepared by simply heating and stirring various molar ratios of N-isopropylacrylamide (NIPAM), acrylamide (AAm) and 2-hydroxyethyl methacrylate (HEMA). The structural and thermal properties of the resulting mixtures are evaluated by 1D and 2D NMR spectroscopy as well as differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). UV photocuring kinetics of the PE mixtures is evaluated via in situ photo-DSC and photorheology measurements. The PE mixtures cure rapidly and display storage moduli that are orders of magnitude greater than equivalent copolymers cured in an aqueous medium. The versatility of these PE systems is demonstrated through the addition of a photoswitchable spiropyran acrylate monomer, as well as applying the PE formulation as a stereolithography (SLA)-based 3D printing resin. Due to the hydrogen-bonding network in PE systems, 3D printing of the eutectic resin is possible in the absence of crosslinkers. The addition of a RAFT agent to reduce average polymer chain length enables 3D printing of materials which retain their shape and can be dissolved on demand in appropriate solvents., (© 2024 The Authors. Macromolecular Rapid Communications published by Wiley‐VCH GmbH.)

Photocontrolled deprotection of specific functional groups has garnered significant interest over the past two decades. Notably, the selective deprotection of distinct groups based on wavelength has emerged as a prominent focus in recent research. The achievement of this objective has primarily involved the utilization of linker-based bichromophoric systems and diverse cocktail mixtures of photoresponsive protecting groups (PRPGs), each responsive to varying wavelengths of light. Herein, we present the first wavelength-selective monochromophoric system based on a hydroxanthene moiety, enabling the wavelength-selective release of two distinct functionalities under 450 and 600 nm light, respectively. The mechanism of the wavelength-selective photodegradation was thoroughly investigated by 1 H NMR, UV-vis, and fluorescence spectroscopy, suggesting a proton-coupled electron transfer mechanism in the first photorelease step and electron transfer based arylmethyl type of photorelease in the second step. The utility of the xanthene-based wavelength-selective PRPGs was demonstrated in the multistep degradation of microparticles and dual-color tuning of polymer chain architecture, thus opening an avenue to design advanced photoreactive wavelength-controlled systems for applications in soft matter materials.

The wavelength-by-wavelength resolved photoreactivity of two photo-caged carboxylic acids, i. e. 7-(diethylamino)-coumarin- and 3-perylene-modified substrates, is investigated via photochemical action plots. The observed wavelength-dependent reactivity of the chromophores is contrasted with their absorption profile. The photochemical action plots reveal a remarkable mismatch between the maximum reactivity and the absorbance. Through the action plot data, the study is able to uncover photochemical reactivity maxima at longer and shorter wavelengths, where the molar absorptivity of the chromophores is strongly reduced. Finally, the laser experiments are translated to light emitting diode (LED) irradiation and show efficient visible-light-induced release in a near fully wavelength-orthogonal, sequence-independent fashion (λ LED1  = 405 nm, λ LED2  = 505 nm) with both chromophores in the same reaction solution. The herein pioneered wavelength orthogonal release systems open an avenue for releasing two different molecular cargos with visible light in a fully orthogonal fashion., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Thioindigos are visible light responsive photoswitches with excellent spatial control over the conformational change between their trans- and cis- isomers. However, they possess limited solubility in all conventional organic solvents and polymers, hindering their application in soft matter materials. Herein, we introduce a strategy for the covalent insertion of thioindigo units into polymer main chains, enabling thioindigos to function within crosslinked polymeric hydrogels. We overcome their solubility issue by developing a thioindigo bismethacrylate linker able to undergo radical initiated thiol-ene reaction for step-growth polymerization, generating indigo-containing polymers. The optimal wavelength for the reversible trans-/cis- isomerisation of thioindigo was elucidated by constructing a detailed photochemical action plot of their switching efficiencies at a wide range of monochromatic wavelengths. Critically, indigo-containing polymers display significant photoswitching of the materials' optical and physical properties in organic solvents and water. Furthermore, the photoswitching of thioindigo within crosslinked structures enables visible light induced modulation of the hydrogel stiffness. Both the thioindigo-containing hydrogels and photoswitching processes are non-toxic to cells, thus offering opportunities for advanced applications in soft matter materials and biology-related research., (© 2023. The Author(s).)

Microgels are microscale particles of hydrogel that can be laden with cells and used to create macroporous tissue constructs. Their ability to support cell-ECM and cell-cell interactions, along with the high levels of nutrient and metabolite exchange facilitated by their high surface area-to-volume ratio, means that they are attracting increasing attention for a variety of tissue regeneration applications. Here, we present methods for fabricating and modifying the structure of microfluidic devices using commonly available laboratory consumables including pipet tips and PTFE and silicon tubing to produce microgels. Different microfluidic devices realized the controlled generation of a wide size range (130-800 μm) of microgels for cell encapsulation. Subsequently, we describe the process of encapsulating mesenchymal stromal cells in microgels formed by photo-cross-linking of gelatin-norbornene and PEG dithiol. The introduced pipet-based chip offers simplicity, tunability, and versatility, making it easily assembled in most laboratories to effectively produce cell-laden microgels for various applications in tissue engineering.

We map the photochemical reactivity of two chromophores-a pyrene-chalcone and a methylene blue protected amine-from a one-pot reaction mixture based on their dynamic absorptivity changes upon light exposure, constructing a dual action plot. We employ the action plot data to determine a pathlength-independent λ-orthogonality window, allowing the orthogonal folding of distinct polymer chains into single chain nano-particles (SCNPs) from the same reaction mixture.

We introduce a class of single-chain nanoparticles (SCNPs) that respond to visible light (λ max =415 nm) with complete unfolding from their compact structure into linear chain analogues. The initial folding is achieved by a simple esterification reaction of the polymer backbone constituted of acrylic acid and polyethylene glycol carrying monomer units, introducing bimane moieties, which allow for the photochemical unfolding, reversing the ester-bond formation. The compaction and the light driven unfolding proceed cleanly and are readily followed by size exclusion chromatography (SEC) and diffusion ordered NMR spectroscopy (DOSY), monitoring the change in the hydrodynamic radius (R H ). Importantly, the folding reaction and the light-induced unfolding are reversible, supported by the high conversion of the photo cleavage. As the unfolding reaction occurs in aqueous systems, the system holds promise for controlling the unfolding of SCNPs in biological environments., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Light degradable polymers hold significant promise in a wide range of applications including the fabrication of optically recyclable materials, responsive coatings and adhesives, and controlled drug delivery. Here, we report the synthesis of polyurethanes that can be degraded under irradiation of visible light (≤450 nm) from commercial LED (3-15 W) light sources. The photolysis occurs in an aqueous environment via photocleavage of an acridine moiety incorporated within the backbone of the polymer chains. Analysis of the quantum yield as a function of wavelength reveals highly efficient photoreactivity at up to 440 nm activation, which is red-shifted compared to the UV-vis absorbance of the chromophore. The potential of our chemical system in biomaterials is demonstrated by the fabrication of an in situ forming hydrogel that can be degraded by visible light.

We introduce a visible light-driven ( λ max = 451 nm) photo-chemical strategy for labelling of DNA in living HeLa cells via the [2+2] cycloaddition of a styrylquinoxaline moiety, which we incorporate into both the DNA and the fluorescent label. Our methodology offers advanced opportunities for the mild remote labelling of DNA in water while avoiding UV light activation.

We pioneer the photochemical generation of single chain nanoparticles (SCNPs) at the to-date mildest reported wavelength of 625 nm by exploiting the photochemical uncaging of methylene blue protected amines. The protected amines are tethered to polymers prepared via reversible addition-fragmentation chain transfer (RAFT) polymerisation, and subsequently undergo intrachain crosslinking by amide formation.

Photochemical ligation has become an indispensable tool for applications that require spatially addressable functionalisation, both in biology and materials science. Interestingly, a number of photochemical ligations result in fluorescent products, enabling a self-reporting function that provides almost instantaneous visual feedback of the reaction's progress and efficiency. Perhaps no other chemical reaction system allows control in space and time to the same extent, while concomitantly providing inherent feedback with regard to reaction success and location. While photoactivable fluorescent properties have been widely used in biology for imaging purposes, the expansion of the array of photochemical reactions has further enabled its utility in soft matter materials. Herein, we concisely summarise the key developments of fluorogenic-forming photoligation systems and their emerging applications in both biology and materials science. We further summarise the current challenges and future opportunities of exploiting fluorescent self-reporting reactions in a wide array of chemical disciplines., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Chimeric antigen receptor (CAR-) T cells are revolutionizing cancer treatment, as a direct result of their clinical impact on the treatment of hematological malignancies. However for solid tumors, CAR-T cell therapeutic efficacy remains limited, primarily due to the complex immunosuppressive tumor microenvironment, inefficient access to tumor cells and poor persistence of the killer cells. In this in vitro study, an injectable, gelatin-based micro-hydrogel system that can encapsulate and deliver effective CAR-T therapy is investigated. CAR-T cells targeting TAG-72, encapsulated in these microgels possessed high viability (> 87%) after 7 days, equivalent to those grown under normal expansion conditions, with retention of the T cell phenotype and functionality. Microgel recovered CAR-T cells demonstrated potent on-target cytotoxicity against human ovarian cancer in vitro and on three-dimensional tumor spheroids, by completely eliminating tumor cells. The gelatin-based micro-hydrogels have the potential to serve as carrier systems to augment CAR-T immunotherapeutic treatment of solid tumors., (Copyright © 2022. Published by Elsevier Inc.)

The additive manufacturing technique direct laser writing (DLW), also known as two-photon laser lithography, is becoming increasingly established as a technique capable of fabricating functional 3D microstructures. Recently, there has been an increasing effort to impart microstructures fabricated using DLW with advanced functionalities by introducing responsive chemical entities into the underpinning photoresists. Herein, a novel photoresist based on the photochemistry of the bimane group is introduced that can be degraded upon exposure to very mild conditions, requiring only water and visible light (λ max  = 415-435 nm) irradiation. The degradation of the microstructures is tracked and quantified using AFM measurements of their height. The influence of the writing parameters as well as the degradation conditions is investigated, unambiguously evidencing effective visible light degradation in aqueous environments. Finally, the utility of the photodegradable resist system is demonstrated by incorporating it into multimaterial 3D microstructures, serving as a model for future applications., (© 2022 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

We introduce a visible light-induced Staudinger-Bertozzi ligation via photo-uncaging of a triphenylphosphine moiety with a photolabile coumarin derivative. Our action plot study examines the conversion as the function of wavelength, revealing that the uncaging process and Staudinger reaction can be triggered by green light ( λ < 550 nm). We further demonstrate the applicability of our approach in materials science via endgroup modification of water soluble poly(ethylene glycol) and green light-induced patterning of a solid substrate.

We introduce a photochemical bond forming system, where two colours of light are required to trigger covalent bond formation. Specifically, we exploit a visible light cis/trans isomerization of chlorinated azobenzene, which can only undergo reaction with a photochemically generated ketene in its cis state. Detailed photophysical mapping of the reaction efficiencies at a wide range of monochromatic wavelengths revealed the optimum irradiation conditions. Subsequent small molecule and polymer ligation experiments illustrated that only the application of both colours of light affords the reaction product. We further extend the functionality to a photo reversible ketene moiety and translate the concept into material science. The presented reaction system holds promise to be employed as a two-colour resist., (© 2022. The Author(s).)

The ability of light to remotely control the properties of soft matter materials in a dynamic fashion has fascinated material scientists and photochemists for decades. However, only recently has our ability to map photochemical reactivity in a finely wavelength resolved fashion allowed for different colors of light to independently control the material properties of polymer networks with high precision, driven by monochromatic irradiation enabling orthogonal reaction control. The current concept article highlights the progress in visible light-induced photochemistry and explores how it has enabled the design of polymer networks with dynamically adjustable properties. We will explore current applications ranging from dynamic hydrogel design to the light-driven adaptation of 3D printed structures on the macro- and micro-scale. While the alternation of mechanical properties via remote control is largely reality for soft matter materials, we herein propose the next frontiers for adaptive properties, including remote switching between conductive and non-conductive properties, hydrophobic and hydrophilic surfaces, fluorescent or non-fluorescent, and cell adhesive vs. cell repellent properties., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Herein, we introduce the wavelength-orthogonal crosslinking of hydrogel networks using two red-shifted chromophores, i.e. acrylpyerene (AP, λ activation =410-490 nm) and styrylpyrido[2,3-b]pyrazine (SPP, λ activation =400-550 nm), able to undergo [2+2] photocycloaddition in the visible-light regime. The photoreactivity of the SPP moiety is pH-dependent, whereby an acidic environment inhibits the cycloaddition. By employing a spiropyran-based photoacid generator with suitable absorption wavelength, we are able to restrict the activation wavelength of the SPP moiety to the green light region (λ activation =520-550 nm), enabling wavelength-orthogonal activation of the AP group. Our wavelength-orthogonal photochemical system was successfully applied in the design of hydrogels whose stiffness can be tuned independently by either green or blue light., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Predicting wavelength-dependent photochemical reactivity is challenging. Herein, we revive the well-established tool of measuring action spectra and adapt the technique to map wavelength-resolved covalent bond formation and cleavage in what we term "photochemical action plots". Underpinned by tunable lasers, which allow excitation of molecules with near-perfect wavelength precision, the photoinduced reactivity of several reaction classes have been mapped in detail. These include photoinduced cycloadditions and bond formation based on photochemically generated o -quinodimethanes and 1,3-dipoles such as nitrile imines as well as radical photoinitiator cleavage. Organized by reaction class, these data demonstrate that UV/vis spectra fail to act as a predictor for photochemical reactivity at a given wavelength in most of the examined reactions, with the photochemical reactivity being strongly red shifted in comparison to the absorption spectrum. We provide an encompassing perspective of the power of photochemical action plots for bond-forming reactions and their emerging applications in the design of wavelength-selective photoresists and photoresponsive soft-matter materials.

Photoresponsive hydrogels hold key potential in advanced biomedical applications including tissue engineering, regenerative medicine, and drug delivery, as well as intricately engineered functions such as biosensing, soft robotics, and bioelectronics. Herein, the wavelength-dependent degradation of bio-orthogonal poly(ethylene glycol) hydrogels is reported, using three selective activation levels. Specifically, three chromophores are exploited, that is, ortho-nitrobenzene, dimethyl aminobenzene, and bimane, each absorbing light at different wavelengths. By examining their photochemical action plots, the wavelength-dependent reactivity of the photocleavable moieties is determined. The wavelength-selective addressability of individual photoreactive units is subsequently translated into hydrogel design, enabling wavelength-dependent cleavage of the hydrogel networks on-demand. Critically, this platform technology allows for the fabrication of various hydrogels, whose mechanical properties can be fine-tuned using different colors of light to reach a predefined value, according to the chromophore ratios used. The softening is shown to influence the spreading of pre-osteoblastic cells adhering to the gels as a demonstration of their potential utility. Furthermore, the materials and photodegradation processes are non-toxic to cells, making this platform attractive for biomaterials engineering., (© 2021 Wiley-VCH GmbH.)

We introduce a four component Passerini polymerization utilizing sterically bulky isocyanide monomers. Under typical Passerini conditions, bulky isocyanides do not react within standard Passerini reaction timescales (hours). We overcome this challenge via the unique physiochemical conditions present in a vortex fluidic device, reducing the reaction time to 2 h on average. Under these high-shear thin-film conditions, bulky isocyanides are readily incorporated into the multicomponent polymerization without the need of high-pressure or temperature. Finally, we demonstrate that the four component approach using functional cyclic anhydrides allows for post-polymerization modification.

We introduce a highly efficient ligation system based on a visible light-induced rearrangement affording a thiophenol which rapidly undergoes thiol-Michael additions. Unlike conventional light-triggered thiol-ene/yne systems, which rely on the use of photocaged bases/nucleophiles, (organo)-photo catalysts, or radical photoinitiators, our system provides a light-induced reaction in the absence of any additives. The ligation is self-catalyzed via the pyridine mediated deprotonation of the photochemically generated thiophenol. Subsequently, the thiol-Michael reaction between the thiophenol anion and electron deficient alkynes/alkenes proceeds additive-free. Hereby, the underlying photoinduced rearrangement of o -thiopyrinidylbenzaldehyde ( o TPyB) generating the free thiol is described for the first time. We studied the influence of various reactions conditions as well as solvents and substrates. We exemplify our findings in a polymer end group modification and obtained macromolecules with excellent end group fidelity.

Residual microtumors following surgical resection are the major cause for lethal cancer recurrence. However, it remains challenging to completely eliminate these residual microtumors. Here, we report an integrated strategy for image-guided surgical resection of tumors and intraoperative surface-enhanced Raman spectroscopy (SERS) guided thermosurgical elimination of residual microtumors using a "three-in-one" theranostic nanoprobe, termed the Au nanostar-based photoacoustic (PA), SERS, and thermosurgical (starPART) probe. This starPART probe, comprising an Au nanostar core, a Raman molecule layer, and a silica outer layer, draws upon the significant advantages of PA imaging, SERS detection, and photothermal tumor ablation. These prominent features enable preoperative PA imaging for surgical resection of tumors and intraoperative SERS-guided thermosurgery for complete elimination of residual microtumors. In vivo experiments confirm complete eradication of microtumors without local recurrence and with a 100% tumor-free survivability. This work therefore offers a robust platform for real-time intraoperative eradication of residual microtumors with significant improvement of surgical outcomes.

Surface coatings are extensively applied on biomedical devices to provide protection against biofouling and infections. However, most surface coatings prevent both bacteria and cells interactions with the biomaterials, limiting their uses as implants. Furthermore, damage to the surface such as scratches and abrasions can happen during transport and clinical usage, resulting in the loss of antibacterial property. In this work, we introduce an efficient method to fabricate stable anti-infective and self-healable multilayer coatings on stainless steel surface via a three-step procedue. Firstly, modified polyethyleneimine (PEI) and poly(acrylic acid) (PAA), both contain pendant furan groups, were deposited on the surface using Layer-by-Layer (LbL) self-assembly technique. Secondly, the polymer layers were cross-linked, via Diels-Alder cycloaddition, using a bismaleimide poly(ethylene glycol) linker, to enhance the stability of the coatings. Thirdly, the Diels-Alder adduct was utilised in the thiol-ene click reaction for post-modification of the coatings, which allowed for the grafting of antimicrobial poly(hexamethylene biguanide) (PHMB) and ε-poly(lysine) (EPL). The resultant multilayer coatings not only exhibited rapid self-healing property, with complete scratch closure within 30 min, but also demonstrated effective antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In addition, biofouling of bovine serum albumin was found to be inhibited on the coated surfaces. Furthermore, these coatings showed no toxicity effect towards seeded osteoblastic cells (MC3T3-E1) and evidence of anti-inflamatory activity when tested against macrophage cell line U-937. Our coating method thus represents an effective strategy for the anti-infective protection of biomedical-devices having direct contact with tissues., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Light-mediated polymer cross-linking is frequently employed for the preparation of hydrogels for biomedical applications. However, most photopolymerization processes require activation by UV light or short wavelength visible light, which are highly absorbed by skin and tissue, limiting their uses in transdermal initiation. Herein, we introduce red light-enabled oxime ligation by the in situ photogeneration of aldehydes, which rapidly react with hydroxylamines. We demonstrate efficient polymer cross-linking behind a dermal tissue model by red light initiation. Optimization of the photopolymerization conditions allows for 3D encapsulation of human foreskin fibroblasts with good cell viability postencapsulation.

Microgels are emerging as an outstanding platform for tissue regeneration because they overcome issues associated with conventional bulk/macroscopic hydrogels such as limited cell-cell contact and cell communication and low diffusion rates. Owing to the enhanced mass transfer and injectability via a minimally invasive procedure, these microgels are becoming a promising approach for bone regeneration applications. Nevertheless, there still remains a huge gap between the understanding of how the hydrogel matrix composition can influence cell response and overall tissue formation when switching from bulk formats to microgel format, which is often neglected or rarely studied. Here, we fabricated polyethylene glycol-based microgels and bulk hydrogels incorporating gelatin and hyaluronic acid (HA), either individually or together, and assessed the impact of both hydrogel composition and format upon the osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hBMSCs). Osteogenesis was significantly greater in microgels than bulk hydrogels for both gelatin alone (Gel) and gelatin HA composite (Gel:HA) hydrogels, as determined by the expression of Runt-related transcription factor (Runx2) and alkaline phosphatase (ALP) genes and mineral deposition. Interestingly, Gel and Gel:HA hydrogels behaved differently between bulk and microgel format. In bulk format, overall osteogenic outcomes were better in Gel:HA hydrogels, but in microgel format, while the level of osteogenic gene expression was equivalent between both compositions, the degree of mineralization was reduced in Gel:HA microgels. Investigation into the affinity of hydroxyapatite for the different matrix compositions indicated that the decreased mineralization of Gel:HA microgels was likely due to a low affinity of hydroxyapatite to bind to HA and support mineral deposition, which has a greater impact on microgels than bulk hydrogels. Together, these findings suggest that both hydrogel composition and format can determine the success of tissue formation and that there is a complex interplay of these two factors on both cell behavior and matrix deposition. This has important implications for tissue engineering, showing that hydrogel composition and geometry must be evaluated together when optimizing conditions for cell differentiation and tissue formation.

Photochemical reactions are a powerful tool in (bio)materials design due to the spatial and temporal control light can provide. To extend their applications in biological setting, the use of low-energy, long wavelength light with high penetration propertiesis required. Further regulation of the photochemical process by additional stimuli, such as pH, will open the door for construction of highly regulated systems in nanotechnology- and biology-driven applications. Here we report the green light induced [2+2] cycloaddition of a halochromic system based on a styrylquinoxaline moiety, which allows for its photo-reactivity to be switched on and off by adjusting the pH of the system. Critically, the [2+2] photocycloaddition can be activated by green light (λ up to 550 nm), which is the longest wavelength employed to date in catalyst-free photocycloadditions in solution. Importantly, the pH-dependence of the photo-reactivity was mapped by constant photon action plots. The action plots further indicate that the choice of solvent strongly impacts the system's photo-reactivity. Indeed, higher conversion and longer activation wavelengths were observed in water compared to acetonitrile under identical reaction conditions. The wider applicability of the system was demonstrated in the crosslinking of an 8-arm PEG to form hydrogels (ca. 1 cm in thickness) with a range of mechanical properties and pH responsiveness, highlighting the potential of the system in materials science.

T cells are predominantly produced by the thymus and play a significant role in maintaining our adaptive immune system. Physiological involution of the thymus occurs gradually with age, compromising naive T cell output, which can have severe clinical complications. Also, T cells are utilized as therapeutic agents in cancer immunotherapies. Therefore, there is an increasing need for strategies aimed at generating naive T cells. The majority of in vitro T cell generation studies are performed in two-dimensional (2D) cultures, which ignore the physiological thymic microenvironment and are not scalable; therefore, we applied a new three-dimensional (3D) approach. Here, we use a gelatin-based 3D microgel system for T lineage induction by co-culturing OP9-DL4 cells and mouse fetal-liver-derived hematopoietic stem cells (HSCs). Flow cytometric analysis revealed that microgel co-cultures supported T lineage induction similar to 2D cultures while providing a 3D environment. We also encapsulated mouse embryonic thymic epithelial cells (TECs) within the microgels to provide a defined 3D culture platform. The microgel system supported TEC maintenance and retained their phenotype. Together, these data show that our microgel system has the capacity for TEC maintenance and induction of in vitro T lineage differentiation with potential for scalability.

The encapsulation of cells into microgels is attractive for applications in tissue regeneration. While cells are protected against shear stress during injection, the assembly of microgels after injection into a tissue defect also forms a macroporous scaffold that allows effective nutrient transport throughout the construct. However, in most of current strategies that form microgel-based macroporous scaffold or higher-order structures, cells are seeded during or post the assembly process and not microencapsulated in situ. The objective of this study is to investigate the chondrogenic phenotype of microencapsulated fetal chondrocytes in a biocompatible, assembled microgel system vs. bulk gels and to test the stability of the constructs in vivo. Here, we demonstrate that cell microencapsulation leads to increased expression of cartilage-specific genes in a TGF-β1-dependent manner. This correlates, as shown by histological staining, with the ability of microencapsulated cells to deposit cartilaginous matrix after migrating to the surface of the microgels, while keeping a macroscopic granular morphology. Implantation of precultured scaffolds in a subcutaneous mouse model results in vessel infiltration in bulk gels but not in assembled microgels, suggesting a higher stability of the matrix produced by the cells in the assembled microgel constructs. The cells are able to remodel the microgels as demonstrated by the gradual disappearance of the granular structure in vivo. The biocompatible microencapsulation and microgel assembly system presented in this article therefore hold great promise as an injectable system for cartilage repair.

Photochemical ligation is important in biomaterials engineering for spatiotemporal control of biochemical processes. Such reactions however generally require activation by high energy UV or short wavelength blue light, which can limit their use as a consequence of the potential of these high energy light sources to damage living cells. Herein, we present an additive-free, biocompatible, chemical ligation triggered by mild visible light. BODIPY dyes with a pendant thioether attached at the meso-position undergo photolysis of the [C-S] bond under green light (λ=530 nm) excitation, producing an ion pair intermediate that can react specifically with a propiolate group. The utility of this photochemical ligation in materials science is demonstrated by the fabrication of hydrogels with specific architectures, photo-immobilization of biomacromolecules, and live cell encapsulation within a hydrogel scaffold., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrogels are attractive candidates for use in tissue-engineering and the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports demonstrate that mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, using a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we show that particle size, charge, and hydrogel mechanical properties all influence the diffusion of embedded transfection agent complexes. By incorporating both MSCs and transfection agents into the hydrogels we demonstrate successful in situ transfection of encapsulated MSCs. Comparing the efficacy of pre- and in situ transfection of miR-100-5p/miR-143-3p on the osteogenic capacity of hydrogel-encapsulated MSCs, our data demonstrates superior mineralisation and osteogenic gene expression following in situ transfections. Overall, we demonstrate a simple, one-pot system for in situ transfection of miRNAs to enhance MSC osteogenic potential and thus demonstrates significant promise to improve the efficiency of MSC differentiation in hydrogels for bone tissue-engineering applications. STATEMENT OF SIGNIFICANCE: Mesenchymal stromal cells (MSCs) are sensitive to cues from their surrounding microenvironment. Osteogenesis is enhanced in MSCs grown on stiffer substrates, but this is limited when using hydrogels for bone tissue-engineering. Modulating pro-osteogenic genes with mechanosensitive microRNAs (miRNAs) represents a potential tool to overcome this challenge. Here we report a hydrogel platform to deliver miRNAs to encapsulated MSCs. We characterise effects of hydrogel composition and transfection agent type on their mobility and transfection efficiency, demonstrating successful in situ transfection of MSCs and showing that miRNAs can significantly enhance osteogenic mineral deposition and marker gene expression. This system was simpler and more effective than conventional 2D transfection prior to encapsulation and therefore holds promise to improve MSC differentiation in bone tissue-engineering., (Copyright © 2019. Published by Elsevier Ltd.)

On-demand photo-uncaging of reactive thiols have been employed in engineering biomaterial scaffolds for regulation of cellular activities. A drawback of the current photo-uncaging chemistry is the utilization of high energy UV light or 2-photon laser light, which may be harmful to cells and cause undesired side reactions within the biological environment. We introduce an effective approach for the caging of thiol using monobromobimane, which can be removed under irradiation of light at λ = 420 nm and in the presence of electrophiles, such as acrylate, propiolate and maleimide, for trapping of the newly release thiol. This chemical approach can be used in visible light-induced polymer coupling and cross-linking for the preparation of cell-laden hydrogels.

Current clinical approaches to treat articular cartilage degeneration provide only a limited ability to regenerate tissue with long-term durability and functionality. In this application, injectable bulk hydrogels and microgels containing stem cells can provide a suitable environment for tissue regeneration. However insufficient cell-cell interactions, low differentiation efficiency and poor tissue adhesion hinder the formation of high-quality hyaline type cartilage. Here, we have designed a higher order tissue-like structure using injectable cell-laden microgels as the building blocks to achieve human bone marrow-derived mesenchymal stem cell (hBMSC) long-term maintenance and chondrogenesis. We have demonstrated that a 4-arm poly(ethylene glycol)-N-hydroxysuccinimide (NHS) crosslinker induces covalent bonding between the microgel building blocks as well as the surrounding tissue mimic. The crosslinking process assembles the microgels into a 3D construct and preserves the viability and cellular functions of the encapsulated hBMSCs. This assembled microgel construct encourages upregulation of chondrogenic markers in both gene and glycosaminoglycan (GAG) expression levels. In addition, the regenerated tissue in the assembled microgels stained positively with Alcian blue and Safranin O exhibiting unique hyaline-like cartilage features. Furthermore, the immunostaining showed a favourable distribution and significantly higher content of type II collagen in the assembled microgels when compared to both the bulk hydrogel and pellet cultures. Collectively, this tissue adhesive hBMSC-laden microgel construct provides potential clinical opportunities for articular cartilage repair and other applications in regenerative medicine., Statement of Significance: A reliable approach to reconstruct durable and fully functional articular cartilage tissue is required for effective clinical therapies. Here, injectable hydrogels together with cell-based therapies offer new treatment strategies in cartilage repair. For effective cartilage regeneration, the injectable hydrogel system needs to be bonded to the surrounding tissue and at the same time needs to be sufficiently stable for prolonged chondrogenesis. In this work, we utilised injectable hBMSC-laden microgels as the building blocks to create an assembled construct via N-hydroxysuccinimide-amine coupling. This crosslinking process also allows for rapid bonding between the assembled microgels and a surrounding tissue mimic. The resultant assembled microgel-construct provides both a physically stable and biologically dynamic environment for hBMSC chondrogenesis, leading to the production of a mature hyaline type cartilage structure., (Copyright © 2018 Acta Materialia Inc. All rights reserved.)

Myocardial tissue engineering is a promising therapy for myocardial infarction recovery. The success of myocardial tissue engineering is likely to rely on the combination of cardiomyocytes, prosurvival regulatory signals, and a flexible biomaterial structure that can deliver them. In this study, poly(glycerol sebacate) (PGS), which exhibits stable elasticity under repeated tensile loading, was engineered to provide physical features that aligned cardiomyocytes in a similar manner to that seen in native cardiac tissue. In addition, a small molecule mimetic of brain derived neurotrophic factor (BDNF) was polymerized into the PGS to achieve a continuous and steady release. Micropatterning of PGS elastomers increased cell alignment, calcium transient homogeneity, and cell connectivity. The intensity of the calcium transients in cardiomyocytes was enhanced when cultured on PGS which released a small molecule BDNF mimetic. This study demonstrates that robust micropatterned elastomer films are a potential candidate for the delivery of functional cardiomyocytes and factors to the injured or dysfunctional myocardium, as well as providing novel in vitro platforms to study cardiomyocyte physiology.

Reversible photocycloaddition reactions have previously been employed in chemical cross-linking for the preparation of biomaterial scaffolds. However, the processes require activation by high-energy UV light, rendering them unsuitable for modification in biological environments. Here we demonstrate that the [2 + 2] photocycloaddition of styrylpyrene can be activated by visible light at λ = 400-500 nm, enabling rapid and effective conjugation and cross-linking of poly(ethylene glycol) (PEG) in water and under mild irradiation conditions ( I = 20 mW cm -2 ). Notably, the reversion of the cycloaddition can be triggered by low-energy UV light at λ = 340 nm, which allows for efficient cleavage of the dimer adduct. Using this wavelength-gated reversible photochemical reaction we are able to prepare PEG hydrogels and modulate their mechanical properties in a bidirectional manner. We also demonstrate healing of the fractured hydrogel by external light triggers. Furthermore, we show that human mesenchymal stem cells can be encapsulated within the gels with high viability post encapsulation. This photochemical approach is therefore anticipated to be highly useful in studies of cell mechanotransduction, with relevance to disease progression and tissue regeneration.

Tetrazine-norbornene ligation has previously been applied in bioorthognal polymer crosslinking to form hydrogels suitable for 3D cell culture. However, the tetrazine group is prone to reduction by the free thiol in a biological environment, reducing the crosslinking efficiency and shortening the storage of tetrazine containing linkers. Here, we introduce a method to form a tetrazine group in situ by catalytic oxidation of the dihydrogen tetrazine using horse radish peroxidase (HRP). Enzymatic oxidation is highly efficient at a low HRP concentration and does not require hydrogen peroxide, allowing for rapid gelation when HRP was added to an aqueous solution of 4-arm PEG dihydrogentetrazine and gelatin norbornene. The storage modulus of the resultant gels can be varied by changing the concentration of the crosslinker, which is in the range of 1.2-3.8 kPa. Human mesenchymal stem cells encapsulated within these gels, with varying stiffness, display varied interactions and morphologies and can be maintained with prolonged culture periods of at least 32 days of 3D culture. The enzymatic activation of tetrazine-norbornene is therefore an attractive addition to the tetrazine ligation that is highly suitable for cell related studies in tissue engineering.

We introduce a click and visible-light triggered unclick approach via thio-bromo reaction and hydroquinone photoreduction/trimethyl lock cleavage for polymer modifications. Both reactions can be carried out in water and at ambient temperature, enabling preparation of bioorthogonal hydrogels for encapsulation and controlled release of various cells.

We introduce an efficient method for the preparation of photolabile polymer linkers to be used in the fabrication of bioorthogonal and photodegradable hydrogels. The versatility of this synthesis strategy allows for incorporation of a series of chromophores responsive to addressable wavelengths of UV and broad spectrum visible light. Consequently, selective release of different cell types from composite hydrogels by user-defined timing can be achieved by irradiating the materials with different wavelengths of light.

Recent developments in photochemistry have introduced new methods to prepare hydrogels initiated by nonharmful light which is essential for encapsulation of cells and bioactive components. However, bioorthogonal photoclick reactions generally requires two components for cross-linking and, in many cases, the formation of a reactive intermediate that may cross-react with nucleophiles in biological media. Here we report the utilization of a visible light triggered dimerization of electron-rich anthracene for polymer cross-linking to form bulk hydrogels and microgels. Incorporation of gelatin within the hydrogel enhanced cell attachment and viability after 7 days of culture and spatiotemporal conjugation of a bioactive component using photochemical dimerization of anthracene was demonstrated. This work therefore introduces a simple yet powerful tool for light modulated bioorthogonal polymer cross-linking, which can be utilized in various bioengineering applications.

Stem cell injections for the treatment of articular cartilage damage are a promising approach to achieve tissue regeneration. However, this method is encumbered by high cell apoptosis rates, low retention in the cartilage lesion, and inefficient chondrogenesis. Here, we have used a facile, very low cost-based microfluidic technique to create visible light-cured microgels composed of gelatin norbornene (GelNB) and a poly(ethylene glycol) (PEG) cross-linker. In addition, we have demonstrated that the process enables the rapid in situ microencapsulation of human bone marrow-derived mesenchymal stem cells (hBMSCs) under biocompatible microfluidic-processing conditions for long-term maintenance. The hBMSCs exhibited an unusually high degree of chondrogenesis in the GelNB microgels with chondro-inductive media, specifically toward the hyaline cartilage structure, with significant upregulation in type II collagen expression compared to the bulk hydrogel and "gold standard" pellet culture. Overall, we have demonstrated that these protein-based microgels can be engineered as promising therapeutic candidates for articular cartilage regeneration, with additional potential to be used in a variety of other applications in regenerative medicine.

Swelling of hydrogels leads to a decrease in mechanical performance coupled with complications in solute diffusion. In addition, hydrogel swelling affects patient safety in biomedical applications such as compression of tissue and fluid blockage. A conventional strategy for suppressing swelling is to introduce a thermoresponsive polymer with a lower critical solution temperature (LCST) within the network structure to counter the water uptake at elevated temperature. However, altering the gel's mechanical strength via modification of the network structure often affects the water uptake behavior and thus a nonswelling platform with tunable mechanical properties suitable for various biomedical applications is desirable. In this study we applied the commercially available triblock PEG-PPG-PEG (Pluronic) as a cross-linker for the preparation of nucleophilic thiol-yne click cross-linked hydrogels with suppressed swelling at physiologically relevant temperature. The mechanical properties and degradation rate of these nonswelling hydrogels can be tuned by judicious combinations of the available linkers. The Pluronic linkers can be applied to prepare biologically relevant gelatin based hydrogels with suppressed swelling under physiological conditions that support attachment of fibroblast cells in 2D culture and controlled release of albumin, paving the way for the development of reliable and better performing soft biomaterials.

Synthetic hydrogel materials offer the ability to tune the mechanical properties of the resultant networks by controlling the molecular structure of the polymer precursors. Herein, we demonstrate that the nucleophilic thiol-yne click reaction presents a highly efficient chemistry for forming robust high water content (ca. 90%) hydrogel materials with tunable stiffness and mechanical properties. Remarkably, optimization of the molecular weight and geometry of the poly(ethylene glycol) (PEG) precursors allows access to materials with compressive strength up to 2.4 MPa, which can be repeatedly compressed to >90% stress. Beyond this, we demonstrate the ability to access hydrogels with storage moduli ranging from 0.2 to 7 kPa. Moreover, we also demonstrate that by a simple precursor blending process, we can access intermediate stiffness across this range with minimal changes to the hydrogel structure. These characteristics present the nucleophilic thiol-yne addition as an excellent method for the preparation of hydrogels for use as versatile synthetic biomaterials.

In most synthetic elastomers, changing the physical properties by monomer choice also results in a change to the crystallinity of the material, which manifests through alteration of its mechanical performance. Using organocatalyzed stereospecific additions of thiols to activated alkynes, high-molar-mass elastomers were isolated via step-growth polymerization. The resulting controllable double-bond stereochemistry defines the crystallinity and the concomitant mechanical properties as well as enabling the synthesis of materials that retain their excellent mechanical properties through changing monomer composition. Using this approach to elastomer synthesis, further end group modification and toughening through vulcanization strategies are also possible. The organocatalytic control of stereochemistry opens the realm to a new and easily scalable class of elastomers that will have unique chemical handles for functionalization and post synthetic processing., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications.

Light triggered release of an antibiotic from a click crosslinked hydrogel was developed by conjugating ciprofloxacin through a photo-cleavable linker to the hydrogel network structure. Upon irradiation of the hydrogel material with UV light (365 nm) at low intensity, native ciprofloxacin was released into the surrounding environment and could be detected by HPLC. The antimicrobial activity of the released compound on Staphylococcus aureus was demonstrated.

This communication describes the first application of cycloaddition between an in situ generated nitrile oxide with norbornene leading to a polymer crosslinking reaction for the preparation of poly(ethylene glycol) hydrogels under physiological conditions. Hydrogels with high water content and robust physical strength are readily formed within 2-5 min by a simple two-solution mixing method which allows 3D encapsulation of neuronal cells. This bioorthogonal crosslinking reaction provides a simple yet highly effective method for preparation of hydrogels to be used in bioengineering., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we present a method for the fabrication of in situ forming gelatin and poly(ethylene glycol)-based hydrogels utilizing bioorthogonal, strain-promoted alkyne-azide cycloaddition as the cross-linking reaction. By incorporating nitrobenzyl moieties within the network structure, these hydrogels can be designed to be degradable upon irradiation with low intensity UV light, allowing precise photopatterning. Fibroblast cells encapsulated within these hydrogels were viable at 14 days and could be readily harvested using a light trigger. Potential applications of this new class of injectable hydrogel include its use as a 3D culturing platform that allows the capture and release of cells, as well as light-triggered cell delivery in regenerative medicine.

The use of tough hydrogels as biomaterials is limited as a consequence of time-consuming fabrication techniques, toxic starting materials, and large strain hysteresis under deformation. Herein, we report the simultaneous application of nucleophilic thiol-yne and inverse electron-demand Diels-Alder additions to independently create two interpenetrating networks in a simple one-step procedure. The resultant hydrogels display compressive stresses of 14-15 MPa at 98% compression without fracture or hysteresis upon repeated load. The hydrogel networks can be spatially and temporally postfunctionalized via radical thiylation and/or inverse electron-demand Diels-Alder addition to residual functional groups within the network. Furthermore, gelation occurs rapidly under physiological conditions, enabling encapsulation of human cells.

A water-soluble azide-functionalised chitosan was crosslinked with propiolic acid ester-functional poly(ethylene glycol) using copper-free click chemistry. The resultant hydrogel materials were formed within 5-60 min at 37 °C and resulted in mechanically robust materials with tuneable properties such as swelling, mechanical strength and degradation. Importantly, the hydrogels supported mesenchymal stem cell attachment and proliferation and were also non-toxic to encapsulated cells. As such these studies indicate that the hydrogels have potential to be used as injectable biomaterials for tissue engineering.