Showing papers by "Aránzazu del Campo published in 2020"

4D hydrogel for dynamic cell culture with orthogonal, wavelength-dependent mechanical and biochemical cues

Abstract: Cooperative action of biochemical and biomechanical signals regulates the interactions between cells and the supporting matrix in natural tissues. Herein, we describe a hydrogel for 4D cell culture which allows user-defined stiffening of the cellular environment and presentation of bioadhesive cues in an orthogonal manner using light of different wavelengths. Stiffening of the gel is initiated by VIS light, while activation of the biochemical function is triggered by UV light. We demonstrate the versatility of this system by triggering, directing and/or hindering cell migration from spheroids based on photoactivated stiffening or integrin-binding to the hydrogels. This material allows in situ and independent manipulation of the physicochemical cues in the cellular microenvironment in vitro, and could eventually be extended to in vivo.

Glycerylphytate as an ionic crosslinker for 3D printing of multi-layered scaffolds with improved shape fidelity and biological features

Abstract: The fabrication of intricate and long-term stable 3D polymeric scaffolds by a 3D printing technique is still a challenge. In the biomedical field, hydrogel materials are very frequently used because of their excellent biocompatibility and biodegradability, however the improvement of their processability and mechanical properties is still required. This paper reports the fabrication of dual crosslinked 3D scaffolds using a low concentrated (<10 wt%) ink of gelatin methacryloyl (GelMA)/chitosan and a novel crosslinking agent, glycerylphytate (G1Phy) to overcome the current limitations in the 3D printing field using hydrogels. The applied methodology consisted of a first ultraviolet light (UV) photopolymerization followed by a post-printing ionic crosslinking treatment with G1Phy. This crosslinker provides a robust framework and avoids the necessity of neutralization with strong bases. The blend ink showed shear-thinning behavior and excellent printability in the form of a straight and homogeneous filament. UV curing was undertaken simultaneously to 3D deposition, which enhanced precision and shape fidelity (resolution ≈150 μm), and prevented the collapse of the subsequent printed layers (up to 28 layers). In the second step, the novel G1Phy ionic crosslinker agent provided swelling and long term stability properties to the 3D scaffolds. The multi-layered printed scaffolds were mechanically stable under physiological conditions for at least one month. Preliminary in vitro assays using L929 fibroblasts showed very promising results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers (i.e. TPP). We envision that the proposed combination of the blend ink and 3D printing approach can have widespread applications in the regeneration of soft tissues.

3D Printing of a Reactive Hydrogel Bio-Ink Using a Static Mixing Tool

Abstract: Hydrogel-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.

Thiol-Methylsulfone-Based Hydrogels for 3D Cell Encapsulation

Abstract: Thiol-maleimide and thiol-vinylsulfone cross-linked hydrogels are widely used systems in 3D culture models, in spite of presenting uncomfortable reaction kinetics for cell encapsulation: too fast (seconds for thiol-maleimide) or too slow (minutes-hours for thiol-vinylsulfone). Here, we introduce the thiol-methylsulfone reaction as alternative cross-linking chemistry for cell encapsulation, particularized for PEG-hydrogels. The thiol-methylsulfone reaction occurs at high conversion and at intermediate reaction speed (seconds-minutes) under physiological pH range. These properties allow easy mixing of hydrogel precursors and cells to render homogeneous cell-laden gels at comfortable experimental time scales. The resulting hydrogels are cytocompatible and show comparable hydrolytic stability to thiol-vinylsulfone gels. They allow direct bioconjugation of thiol-derivatized ligands and tunable degradation kinetics by cross-linking with degradable peptide sequences. 3D cell culture of two cell types, fibroblasts and human umbilical vein endothelial cells (HUVECs), is demonstrated.

Photodegradable Hydrogels for Cell Encapsulation and Tissue Adhesion.

Abstract: Hydrogels for wound management and tissue gluing applications have to adhere to tissues for a given time scale and then disappear, either by removal from the skin or by slow degradation for applications inside the body. Advanced wound management materials also envision the encapsulation of therapeutic drugs or cells to support the natural healing process. The design of hydrogels that can fulfill all of these properties with minimal chemical complexity, a stringent condition to favor transfer into a real medical device, is challenging. Herein, we present a hydrogel design with a moderate structural complexity that fulfills a number of relevant properties for wound dressing: it can form in situ and encapsulate cells, it can adhere to tissues, and it can be degraded on demand by light exposure under cytocompatible conditions. The hydrogels are based on starPEG macromers terminated with catechol groups as cross-linking units and contain intercalated photocleavable nitrobenzyl triazole groups. Hydrogels are formed under mild conditions (N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer with 9-18 mM sodium periodate as the oxidant) and are compatible with encapsulated cells. Upon light irradiation, the cleavage of the nitrobenzyl group mediates depolymerization, which enables the on-demand release of cells and debonding from tissues. The molecular design and obtained properties reported here are interesting for the development of advanced wound dressings and cell therapies and expand the range of functionality of current alternatives.

Label-Free Imaging of Cholesterol Assemblies Reveals Hidden Nanomechanics of Breast Cancer Cells.

Abstract: Tumor cells present profound alterations in their composition, structural organization, and functional properties. A landmark of cancer cells is an overall altered mechanical phenotype, which so far are linked to changes in their cytoskeletal regulation and organization. Evidence exists that the plasma membrane (PM) of cancer cells also shows drastic changes in its composition and organization. However, biomechanical characterization of PM remains limited mainly due to the difficulties encountered to investigate it in a quantitative and label-free manner. Here, the biomechanical properties of PM of a series of MCF10 cell lines, used as a model of breast cancer progression, are investigated. Notably, a strong correlation between the cell PM elasticity and oncogenesis is observed. The altered membrane composition under cancer progression, as emphasized by the PM-associated cholesterol levels, leads to a stiffening of the PM that is uncoupled from the elastic cytoskeletal properties. Conversely, cholesterol depletion of metastatic cells leads to a softening of their PM, restoring biomechanical properties similar to benign cells. As novel therapies based on targeting membrane lipids in cancer cells represent a promising approach in the field of anticancer drug development, this method contributes to deciphering the functional link between PM lipid content and disease.

Printed Soft Optical Waveguides of PLA Copolymers for Guiding Light into Tissue

Abstract: The application of optical technologies in treating pathologies and monitoring disease states requires the development of soft, minimal invasive and implantable devices to deliver light to tissues inside the body. Here, we present soft and degradable optical waveguides from poly(d,l-lactide) and derived copolymers fabricated by extrusion printing in the desired dimensions and shapes. The obtained optical waveguides propagate VIS to NIR light in air and in tissue at penetration depths of tens of centimeters. Besides, the printed waveguides have elastomeric properties at body temperature and show softness and flexibility in the range relevant for implantable devices in soft organs. Printed waveguides were able to guide light across 8 cm tissue and activate photocleavage chemical reactions in a photoresponsive hydrogel (in vitro). The simplicity and flexibility of the fiber processing method and the optical and mechanical performance of the obtained waveguides exemplify how rational study of medically approved biomaterials can lead to useful inks for printing cost-effective and flexible optical components for potential use in medical contexts.

Near-infrared-light regulated angiogenesis in a 4D hydrogel

Abstract: Light-responsive hydrogels are useful platforms to study cellular responses. Current photosensitive motifs need UV light to be activated, which is intrinsically cytotoxic and has a low penetration depth in tissues. Herein we describe a strategy for near-infrared (NIR) controlled activation of cellular processes (3D cell spreading and angiogenesis) by embedding upconverting nanoparticles (UCNPs) in a hydrogel modified with light-activatable cell adhesive motifs. The UCNPs can convert NIR light (974 nm) into local UV emission and activate photochemical reactions on-demand. Such optoregulation is spatially controllable, dose-dependent and can be performed at different timepoints of the cell culture without appreciable photodamage of the cells. HUVEC cells embedded in this hydrogel can form vascular networks at predefined geometries determined by the irradiation pattern. The penetration depth of NIR light enabled activation of the angiogenesis response through skin tissue with a thickness of 2.5 mm. Our strategy opens a new avenue for 4D cell cultures, with the potential to be extended to dynamically manipulate cell-matrix interactions and derived cellular processes in vivo.

Printability study of metal ion crosslinked PEG-catechol based inks.

Abstract: In this paper we explore the printability of reversible networks formed by catechol functionalized PEG solutions and metal cations (Al3+, Fe3+ or V3+). The printability and shape fidelity were dependent on the ink composition (metal ion type, pH, PEG molecular weight) and printing parameters (extrusion pressure and printing speed). The relaxation time, recovery rate and viscosity of the inks were analyzed in rheology studies and correlated with thermodynamic and ligand exchange kinetic constants of the dynamic bonds and the printing performance (i.e. shape fidelity of the printed structures). The relevance of the relaxation time and ligand exchange kinetics for printability was demonstrated. Cells seeded on the materials crosslinked with Al3+, Fe3+ ions were viable and revealed well-spread morphologies during 7 day culture, indicating the potential of the formulations to be used as inks for cell encapsulation. The proposed dynamic ink design offers significant flexibility for 3D bioprinting, and enables straightforward adjustment of the printable formulation to meet application-specific needs.

Optoregulated force application to cellular receptors using molecular motors

Abstract: Mechanotransduction events in physiological environments are difficult to investigate, in part due to the lack of experimental tools to apply forces to mechanosensitive receptors remotely. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), here we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The lightdriven actuation of the molecular motor is converted in mechanical twisting of the polymer chains, which will in turn effectively pulls on engaged cell membrane receptors (integrins, cadherins) within the illuminated area. Applied forces have the adequate magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate,for the first time, a functionality which at the moment cannot be achieved by any other means.

Macroscopic Self-Evolution of Dynamic Hydrogels to Create Hollow Interiors.

Abstract: A solid-to-hollow evolution in macroscopic structures is challenging in synthetic materials. A fundamentally new strategy is reported for guiding macroscopic, unidirectional shape evolution of materials without compromising the material's integrity. This strategy is based on the creation of a field with a "swelling pole" and a "shrinking pole" to drive polymers to disassemble, migrate, and resettle in the targeted region. This concept is demonstrated using dynamic hydrogels containing anchored acrylic ligands and hydrophobic long alkyl chains. Adding water molecules and ferric ions (Fe3+ ) to induce a swelling-shrinking field transforms the hydrogels from solid to hollow. The strategy is versatile in the generation of various closed hollow objects (for example, spheres, helix tubes, and cubes with different diameters) for different applications.

Anti-correlation of HER2 and focal adhesion complexes in the plasma membrane.

Abstract: Excess presence of the human epidermal growth factor receptor 2 (HER2) as well as of the focal adhesion protein complexes are associated with increased proliferation, migratory, and invasive behavior of cancer cells. A cross-regulation between HER2 and integrin signaling pathways has been found, but the exact mechanism remains elusive. Here, we investigated whether HER2 colocalizes with focal adhesion complexes on breast cancer cells overexpressing HER2. For this purpose, vinculin or talin green fluorescent protein (GFP) fusion proteins, both key constituents of focal adhesions, were expressed in breast cancer cells. HER2 was either extracellularly or intracellularly labeled with fluorescent quantum dots nanoparticles (QDs). The cell-substrate interface was analyzed at the location of the focal adhesions by means of total internal reflection fluorescent microscopy or correlative fluorescence- and scanning transmission electron microscopy. Expression of HER2 at the cell-substrate interface was only observed upon intracellular labeling, and was heterogeneous with both HER2-enriched and -low regions. In contrast to an expected enrichment of HER2 at focal adhesions, an anti-correlated expression pattern was observed for talin and HER2. Our findings suggest a spatial anti-correlation between HER2 and focal adhesion complexes for adherent cells.

Exogenous Supply of Hsp47 Triggers Fibrillar Collagen Deposition in Skin Cell Cultures in Vitro

Abstract: Collagen is a structural protein that provides mechanical stability and defined architectures to skin. In collagen-based skin disorders this stability is lost, either due to mutations in collagens or in the chaperones involved in collagen assembly. This leads to chronic wounds, skin fragility, and blistering. Existing approaches to treat such conditions rely on administration of small molecules to simulate collagen production, like 4-phenylbutyrate (4-PBA) or growth factors like TGF-β. However, these molecules are not specific for collagen synthesis, and result in unsolicited side effects. Hsp47 is a collagen-specific chaperone with a major role in collagen biosynthesis. Expression levels of Hsp47 correlate with collagen deposition. This article explores the stimulation of collagen deposition by exogenously supplied Hsp47 (collagen specific chaperone) to skin cells, including specific collagen subtypes quantification. Here we quantify the collagen deposition level and the types of deposited collagens after Hsp47 stimulation in different in vitro cultures of cells from human skin tissue (fibroblasts NHDF, keratinocytes HaCat and endothelial cells HDMEC) and mouse fibroblasts (L929 and MEF). We find upregulated deposition of fibrillar collagen subtypes I, III and V after Hsp47 delivery. Network collagen IV deposition was enhanced in HaCat and HDMECs, while fibril-associated collagen XII was not affected by the increased intracellular Hsp47 levels. The deposition levels of fibrillar collagen were cell-dependent i.e. Hsp47-stimulated fibroblasts deposited significantly higher amount of fibrillar collagen than Hsp47-stimulated HaCat and HDMECs. A 3-fold enhancement of collagen deposition was observed in fibroblasts upon repeated dosage of Hsp47 within the first 6 days of culture. Our results provide fundamental understanding towards the idea of using Hsp47 as therapeutic protein to treat collagen disorders.

Photodegradable Tissue-Adhesive Hydrogels

Abstract: Hydrogels for wound management and tissue gluing have to adhere to tissue for a given time scale and then disappear, either by removal from the skin or by slow degradation in applications inside the body. Advanced wound management materials also envision the encapsulation of therapeutic drugs or cells to support the natural healing process. The design of hydrogels that can fulfill all these properties with minimal chemical complexity, a stringent condition to favor transfer into a real medical device, is challenging. Herein, we present a hydrogel design with moderate structural complexity that fulfills a number of relevant properties for wound dressing: it can form in situ and encapsulate cells, it can adhere to tissue, and it can be degraded on demand by light exposure under cytocompatible conditions. The hydrogels are based on starPEG macromers terminated with catechol groups as crosslinking units and contain intercalated photocleavable triazole nitrobenzyl groups. Hydrogels are formed under mild conditions (HEPES buffer with 9-18 mM of sodium periodate as oxidant) and are compatible with encapsulated cells. Upon light-irradiation, the cleavage of the nitrobenzyl group mediates depolymerization, which enables on-demand release of cells or debonding from tissue. The molecular design and obtained properties are interesting for the development of advanced wound dressings and cell therapies, and expand the range of functionality of current alternatives.