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- ItemElastomeric Optical Waveguides by Extrusion Printing(Weinheim : Wiley, 2022) Feng, Jun; Zheng, Yijun; Jiang, Qiyang; Włodarczyk‐Biegun, Małgorzata K.; Pearson, Samuel; del Campo, AránzazuAdvances in optogenetics and the increasing use of implantable devices for therapies and health monitoring are driving demand for compliant, biocompatible optical waveguides and scalable methods for their manufacture. Molding, thermal drawing, and dip-coating are the most prevalent approaches in recent literature. Here the authors demonstrate that extrusion printing at room temperature can be used for continuous fabrication of compliant optical waveguides with polydimethylsiloxane (PDMS) core and crosslinked Pluronic F127-diacrylate (Pluronic-DA) cladding. The optical fibers are printed from fluid precursor inks and stabilized by physical interactions and photoinitiated crosslinking in the Pluronic-DA. The printed fibers show optical loss values of 0.13–0.34 dB cm–1 in air and tissue within the wavelength range of 405–520 nm. The fibers have a Young's Modulus (Pluronic cladding) of 150 kPa and can be stretched to more than 5 times their length. The optical loss of the fibers shows little variation with extension. This work demonstrates how printing can simplify the fabrication of compliant and stretchable devices from materials approved for clinical use. These can be of interest for optogenetic or photopharmacology applications in extensible tissues, like muscles or heart.
- 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.
- ItemRegulating bacterial behavior within hydrogels of tunable viscoelasticity(New York : Cold Spring Harbor Laboratory, 2022) Bhusari, Shardul; Sankaran, Shrikrishnan; del Campo, AránzazuEngineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in material’s composition and function. Understanding how the spatial confinement in 3D affects the behavior of the embedded cells is crucial to design and predict ELM’s function, regulate and minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and decreasing plasticity of the matrix, a decrease in colony volumes and an increase in their sphericity is observed. Protein production surprisingly follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that, bacterial mechanosensitivity can be used to control and regulate the composition and function of ELMs by thoughtful design of the encapsulating matrix, and by following design criteria with interesting similarities to those developed for 3D culture of mammalian cells.
- ItemPIEZO1-mediated mechanosensing governs NK cell killing efficiency and infiltration in three-dimensional matrices([Cold Spring Harbor] : Cold Spring Harbor Laboratory (CSHL), 2024) Yanamandra, Archana K.; Zhang, Jingnan; Montalvo, Galia; Zhou, Xiangda; Biedenweg, Doreen; Zhao, Renping; Sharma, Shulagna; Hoth, Markus; Lautenschläger, Franziska; Otto, Oliver; del Campo, Aránzazu; Qu, BinNatural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK cell killing efficiency in 3D is impaired against softened tumor cells, while it is enhanced against stiffened tumor cells. Notably, the durations required for NK cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK cell functions.
- ItemEncapsulation of bacteria in bilayer Pluronic thin film hydrogels: A safe format for engineered living materials(Amsterdam : Elsevier, 2023) Bhusari, Shardul; Kim, Juhyun; Polizzi, Karen; Sankaran, Shrikrishnan; del Campo, AránzazuIn engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.
- 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.
- ItemRegulating Bacterial Behavior within Hydrogels of Tunable Viscoelasticity(Weinheim : Wiley-VCH, 2022) Bhusari, Shardul; Sankaran, Shrikrishnan; del Campo, AránzazuEngineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in material's composition and function. Understanding how the spatial confinement in 3D can regulate the behavior of the embedded cells is crucial to design and predict ELM's function, minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and elastic response to deformation of the matrix, a decrease in colony volumes and an increase in their sphericity are observed. Protein production follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that matrix design can be used to control and regulate the composition and function of ELMs containing microorganisms. Interestingly, design parameters for matrices to regulate bacteria behavior show similarities to those elucidated for 3D culture of mammalian cells.
- ItemPrinted Degradable Optical Waveguides for Guiding Light into Tissue(Weinheim : Wiley-VCH, 2020) Feng, Jun; Zheng, Yijun; Bhusari, Shardul; Villiou, Maria; Pearson, Samuel; del Campo, AránzazuOptogenetics and photonic technologies are changing the future of medicine. To implement light‐based therapies in the clinic, patient‐friendly devices that can deliver light inside the body while offering tunable properties and compatibility with soft tissues are needed. Here extrusion printing of degradable, hydrogel‐based optical waveguides with optical losses as low as 0.1 dB cm−1 at visible wavelengths is described. Core‐only and core‐cladding fibers are printed at room temperature from polyethylene glycol (PEG)‐based and PEG/Pluronic precursors, and cured by in situ photopolymerization. The obtained waveguides are flexible, with mechanical properties tunable within a tissue‐compatible range. Degradation times are also tunable by adjusting the molar mass of the diacrylate gel precursors, which are synthesized by linking PEG diacrylate (PEGDA) with varying proportions of DL‐dithiothreitol (DTT). The printed waveguides are used to activate photochemical and optogenetic processes in close‐to‐physiological environments. Light‐triggered migration of cells in a photoresponsive 3D hydrogel and drug release from an optogenetically‐engineered living material by delivering light across >5 cm of muscle tissue are demonstrated. These results quantify the in vitro performance, and reflect the potential of the printed degradable fibers for in vivo and clinical applications.
- ItemCompliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response(2021) Escolano, Joan-Carles; Taubenberger, Anna V.; Abuhattum, Shada; Schweitzer, Christine; Farrukh, Aleeza; del Campo, Aránzazu; Bryant, Clare E.; Guck, JochenImmune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1b and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-a were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1b and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.
- 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.