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- ItemLight-Regulated Angiogenesis via a Phototriggerable VEGF Peptidomimetic(Weinheim : Wiley-VCH, 2021) Nair, Roshna V.; Farrukh, Aleeza; del Campo, AránzazuThe application of growth factor based therapies in regenerative medicine is limited by the high cost, fast degradation kinetics, and the multiple functions of these molecules in the cell, which requires regulated delivery to minimize side effects. Here a photoactivatable peptidomimetic of the vascular endothelial growth factor (VEGF) that allows the light-controlled presentation of angiogenic signals to endothelial cells embedded in hydrogel matrices is presented. A photoresponsive analog of the 15-mer peptidomimetic Ac-KLTWQELYQLKYKGI-NH2 (abbreviated PQK) is prepared by introducing a 3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl (DMNPB) photoremovable protecting group at the Trp4 residue. This modification inhibits the angiogenic potential of the peptide temporally. Light exposure of PQK modified hydrogels provide instructive cues to embedded endothelial cells and promote angiogenesis at the illuminated sites of the 3D culture, with the possibility of spatial control. PQK modified photoresponsive biomaterials offer an attractive approach for the dosed delivery and spatial control of pro-angiogenic factors to support regulated vascular growth by just using light as an external trigger.
- ItemLighting the Path: Light Delivery Strategies to Activate Photoresponsive Biomaterials In Vivo(Weinheim : Wiley-VCH, 2021) Pearson, Samuel; Feng, Jun; del Campo, AránzazuPhotoresponsive biomaterials are experiencing a transition from in vitro models to in vivo demonstrations that point toward clinical translation. Dynamic hydrogels for cell encapsulation, light-responsive carriers for controlled drug delivery, and nanomaterials containing photosensitizers for photodynamic therapy are relevant examples. Nonetheless, the step to the clinic largely depends on their combination with technologies to bring light into the body. This review highlights the challenge of photoactivation in vivo, and presents strategies for light management that can be adopted for this purpose. The authors’ focus is on technologies that are materials-driven, particularly upconversion nanoparticles that assist in “direct path” light delivery through tissue, and optical waveguides that “clear the path” between external light source and in vivo target. The authors’ intention is to assist the photoresponsive biomaterials community transition toward medical technologies by presenting light delivery concepts that can be integrated with the photoresponsive targets. The authors also aim to stimulate further innovation in materials-based light delivery platforms by highlighting needs and opportunities for in vivo photoactivation of biomaterials. © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH.
- ItemGelation Kinetics and Mechanical Properties of Thiol-Tetrazole Methylsulfone Hydrogels Designed for Cell Encapsulation(Weinheim : Wiley-VCH, 2022) de Miguel‐Jiménez, Adrián; Ebeling, Bastian; Paez, Julieta I.; Fink‐Straube, Claudia; Pearson, Samuel; del Campo, AránzazuHydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.
- ItemA bio-based route to the carbon-5 chemical glutaric acid and to bionylon-6,5 using metabolically engineered Corynebacterium glutamicum(Cambridge : Royal Society of Chemistry, 2018) Rohles, Christina Maria; Gläser, Lars; Kohlstedt, Michael; Gießelmann, Gideon; Pearson, Samuel; del Campo, Aránzazu; Becker, Judith; Wittmann, ChristophIn the present work, we established the bio-based production of glutarate, a carbon-5 dicarboxylic acid with recognized value for commercial plastics and other applications, using metabolically engineered Corynebacterium glutamicum. The mutant C. glutamicum AVA-2 served as a starting point for strain development, because it secreted small amounts of glutarate as a consequence of its engineered 5-aminovalerate pathway. Starting from AVA-2, we overexpressed 5-aminovalerate transaminase (gabT) and glutarate semialdehyde dehydrogenase (gabD) under the control of the constitutive tuf promoter to convert 5-aminovalerate further to glutarate. The created strain GTA-1 formed glutarate as a major product, but still secreted 5-aminovalerate as well. This bottleneck was tackled at the level of 5-aminovalerate re-import. The advanced strain GTA-4 overexpressed the newly discovered 5-aminovalerate importer NCgl0464 and formed glutarate from glucose in a yield of 0.27 mol mol−1. In a fed-batch process, GTA-4 produced more than 90 g L−1 glutarate from glucose and molasses based sugars in a yield of up to 0.70 mol mol−1 and a maximum productivity of 1.8 g L−1 h−1, while 5-aminovalerate was no longer secreted. The bio-based glutaric acid was purified to >99.9% purity. Interfacial polymerization and melt polymerization with hexamethylenediamine yielded bionylon-6,5, a polyamide with a unique structure.
- ItemPhotoactivatable Hsp47: A tool to control and regulate collagen secretion & assembly(ChemRxiv, 2018) Khan, Essak; Sankaran, Shrikrishnan; Paez, Julieta; Muth, Christina; Han, Mitchell; del Campo, AránzazuHsp47 is a chaperone protein with a fundamental role in the folding, stability and intracellular transport of procollagen triple helices. A light-responsive Hsp47 recombinant protein, engineered to control in situ the production and assembly of cellular collagen is here demonstrated. This novel light-driven tool enables unprecedented fundamental studies of collagen biosynthesis and associated diseases.
- ItemThiol-Methylsulfone Based Hydrogels: Enhanced Control on Gelation Kinetics for 3D Cell Encapsulation(Washington, DC : American Chemical Society, 2019) Farrukh, Aleeza; Włodarczyk-Biegun, Malgorzata K.; del Campo, AránzazuHydrogels are useful temporal matrices for cell culture technologies. The successful mixing and encapsulation of cells within the gel requires the selection of efficient and cytocompatible gelation reactions occurring in the minute timescale under physiological conditions. The thiol-methylsulfonyl (MS) chemical reaction is introduced here as a novel chemistry to encapsulate cells in polymeric matrices. Thiol-MS crosslinking does not require a light activation step and can occur within the seconds-to-minutes timescale by adjusting the pH in the physiological range 8.0-6.6. This reaction is cytocompatible and the reaction product is hydrolytically stable in cell culture media up to 4 weeks. Cell encapsulation protocols enabling comfortable handling and yielding homogenous distribution of the embedded cells are described. All these features are relevant for the application of this crosslinking reaction to biomedical scenarios. Finally, this manuscript also compares the performance of thiol-MS hydrogels with the established thiol-maleimide and thiol-vinylsulfone hydrogels. The benefit of thiol-MS crosslinking in terms of control over hydrogelation kinetics is demonstrated.
- Item3D Printing of a Reactive Hydrogel Bio-Ink Using a Static Mixing Tool(Basel : MDPI, 2020) Puertas-Bartolomé, Maria; Włodarczyk-Biegun, Małgorzata K; del Campo, Aránzazu; Vázquez-Lasa, Blanca; San Román, JulioHydrogel-based bio-inks have recently attracted more attention for 3D printing applications in tissue engineering due to their remarkable intrinsic properties, such as a cell supporting environment. However, their usually weak mechanical properties lead to poor printability and low stability of the obtained structures. To obtain good shape fidelity, current approaches based on extrusion printing use high viscosity solutions, which can compromise cell viability. This paper presents a novel bio-printing methodology based on a dual-syringe system with a static mixing tool that allows in situ crosslinking of a two-component hydrogel-based ink in the presence of living cells. The reactive hydrogel system consists of carboxymethyl chitosan (CMCh) and partially oxidized hyaluronic acid (HAox) that undergo fast self-covalent crosslinking via Schiff base formation. This new approach allows us to use low viscosity solutions since in situ gelation provides the appropriate structural integrity to maintain the printed shape. The proposed bio-ink formulation was optimized to match crosslinking kinetics with the printing process and multi-layered 3D bio-printed scaffolds were successfully obtained. Printed scaffolds showed moderate swelling, good biocompatibility with embedded cells, and were mechanically stable after 14 days of the cell culture. We envision that this straightforward, powerful, and generalizable printing approach can be used for a wide range of materials, growth factors, or cell types, to be employed for soft tissue regeneration.
- ItemCytocompatibility Evaluation of PEG-Methylsulfone Hydrogels(Berkeley, CA : University of California, 2023) Trujillo, Sara; Kasper, Jennifer; de Miguel-Jiménez, Adrián; Abt, Britta; Bauer, Alina; Mekontso, Joëlle; Pearson, Samuel; del Campo, AránzazuMethylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide-thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide-thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide-thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.