Dynamische Biomaterialien

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.

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.

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.

A new series of amphiphilic block copolymers has been prepared by ring opening polymerization (ROP) of cyclic carbonates using PEG as initiator. The light responsive unit [7–(diethylaminocoumarin)–4–yl]methyl ester has been introduced by a modular and versatile supramolecular approach, while a reference covalent copolymer has been synthesized for the shake of comparison. Synthesized copolymers showed monomodal narrow distributions and were able to self-assemble into spherical micelles when dispersed in water. UV irradiation allowed us the modification of the self-assemblies morphology, as proved by means of fluorescence spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Both the supramolecular and covalent functionalized block copolymers were able to encapsulate small fluorescent probes as payload and to release them upon UV and NIR irradiation.

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.

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.

The response of cultured cells to the mechanical properties of hydrogel substrates depends ultimately on the response of single crosslinks to external forces exerted at cell attachment points. We prepared hydrogels by co-polymerization of poly(ethylene glycol diacrylate) (PEGDA) and carboxy poly(ethylene glycol) acrylate (ACPEG-COOH) and confirmed fibroblast spreading on the hydrogel after the ACPEG linker was functionalized with the RGD cell adhesive motif. We performed specific force spectroscopy experiments on the same ACPEG linkers in order to probe the mechanics of single cross-links which mediate the cell attachment and spreading. Measurements were performed with tips of an atomic force microscope (AFM) functionalized with streptavidin and ACPEG linkers functionalized with biotin. We compared hydrogels of varying elastic modulus between 4 and 41 kPa which exhibited significant differences in cell spreading. An effective spring constant for the displacement of single cross-links at the hydrogel surface was derived from the distributions of rupture force and molecular stiffness. A factor of ten in the elastic modulus E of the hydrogel corresponded to a factor of five in the effective spring constant k of single crosslinks, indicating a transition in scaling with the mesh size ξ from the macroscopic E ∝ ξ−3 to the molecular k ∝ ξ−2. The quantification of stiffness and deformation at the molecular length scale contributes to the discussion of mechanisms in force-regulated phenomena in cell biology.

Abstract A substrate mimicking the surface topography and temperature sensitivity of skin goosebumps is fabricated. Close-packed arrays of thermoresponsive microgel particles undergo topographical changes in response to temperature changes between 25 and 37 °C, resembling the goosebump structure that human skin develops in response to temperature changes or other circumstances. Specifically, positively charged poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] (PMETAC) brushes serve as an anchoring substrate for negatively charged poly(NIPAm-co-AA) microgels. The packing density and particle morphology can be tuned by brush layer thickness and pH of the microgel suspension. For brush layer thickness below 50 nm, particle monolayers are observed, with slightly flattened particle morphology at pH 3 and highly collapsed particles at pH above 7. Polymer brush films with thickness above 50 nm lead to the formation of particle multilayers. The temperature responsiveness of the monolayer assemblies allows reversible changes in the film morphology, which in turn affects underwater adhesion and friction at 25 and 37 °C. These results are promising for the design of new functional materials and may also serve as a model for biological structures and processes.

Summary In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices.

Abstract HepaRG is a bipotent stem cell line that can be differentiated towards hepatocyte-like and biliary-like cells. The entire cultivation process requires 1 month and relies on the addition of 2% dimethyl sulfoxide (DMSO) to the culture. Our motivation in this research is to differentiate HepaRG cells (progenitor cells and undifferentiated cells) towards hepatocyte-like cells by minimizing the cultivation time and without using DMSO treatment by instead using a microfluidic device combined with the following strategies: (a) comparison of extracellular matrices (matrigel and collagen I), (b) types of flow (one or both sides), and (c) effects of DMSO. Our results demonstrate that matrigel promotes the differentiation of progenitor cells towards hepatocytes and biliary-like cells. Moreover, the frequent formation of HepaRG cell clusters was observed by a supply of both sides of flow, and the cell viability and liver specific functions were influenced by DMSO. Finally, differentiated HepaRG progenitor cells cultured in a microfluidic device for 14 days without DMSO treatment yielded 70% of hepatocyte-like cells with a highly polarized organization that reacted to stimulation with IL-6 to produce C-reactive protein (CRP). This culture model has high potential for investigating cell differentiation and liver pathophysiology research.