Photo-cross-linked and pH-Switchable Soft Polymer Nanocapsules from Polyglycidyl Ethers.

Soft polymer nanocapsules and microgels, which can adapt their shape and, at the same time, sequester and release molecular payloads in response to an external trigger, are a challenging complement to vesicular structures like polymersomes. In this work, we report the synthesis of such capsules by photo-cross-linking of coumarin-substituted polyglycidyl ethers, which we prepared by Williamson etherification of epichlorohydrin (ECH) repeating units with 7-hydroxycoumarin in copolymers with tert-butyl glycidyl ether (tBGE). To control capsule size, we employed the prepolymers in an o/w miniemulsion, where they formed a gel layer at the interface upon irradiation at 365 nm by [2π + 2π] photodimerization of the coumarin groups. Upon irradiation at 254 nm, the reaction could be reversed and the gel wall could be repeatedly disintegrated and rebuilt. We further demonstrated (i) reversible hydrophilization of the gels by hydrolysis of the lactone rings in coumarin dimers as a mechanism to manipulate the permeability of the capsules and (ii) binding functional molecules as amides. Thus, the presented nanogels are remarkably versatile and can be further used as a carrier system.

How Much Physical Guidance is Needed to Orient Growing Axons in 3D Hydrogels?

Directing cells is essential to organize multi-cellular organisms that are built up from subunits executing specific tasks. This guidance requires a precisely controlled symphony of biochemical, mechanical, and structural signals. While many guiding mechanisms focus on 2D structural patterns or 3D biochemical gradients, injectable material platforms that elucidate how cellular processes are triggered by defined 3D physical guiding cues are still lacking but crucial for the repair of soft tissues. Herein, a recently developed anisotropic injectable hybrid hydrogel (Anisogel) contains rod-shaped microgels that orient in situ by a magnetic field and has propelled studying 3D cell guidance. Here, the Anisogel is used to investigate the dependence of axonal guidance on microgel dimensions, aspect ratio, and distance. While large microgels result in high material anisotropy, they significantly reduce neurite outgrowth and thus the guidance efficiency. Narrow and long microgels enable strong axonal guidance with maximal outgrowth including cell sensing over distances of tens of micrometers in 3D. Moreover, nerve cells decide to orient inside the Anisogel within the first three days, followed by strengthening of the alignment, which goes along with oriented fibronectin deposition. These findings demonstrate the potential of the Anisogel to tune structural and mechanical parameters for specific applications.

Synthetic 3D PEG-Anisogel Tailored with Fibronectin Fragments Induce Aligned Nerve Extension.

An enzymatically cross-linked polyethylene glycol (PEG)-based hydrogel was engineered to promote and align nerve cells in a three-dimensional manner. To render the injectable, otherwise bioinert, PEG-based material supportive for cell growth, its mechanical and biochemical properties were optimized. A recombinant fibronectin fragment (FNIII9*-10/12-14) was coupled to the PEG backbone during gelation to provide cell adhesive and growth factor binding domains in close vicinity. Compared to full-length fibronectin, FNIII9*-10/12-14 supports nerve growth at similar concentrations. In a 3D environment, only the ultrasoft 1 w/v% PEG hydrogels with a storage modulus of ∼10 Pa promoted neuronal growth. This gel was used to establish the first fully synthetic, injectable Anisogel by the addition of magnetically aligned microelements, such as rod-shaped microgels or short fibers. The Anisogel led to linear neurite extension and represents a large step in the direction of clinical translation with the opportunity to treat acute spinal cord injuries.

Cell Encapsulation in Soft, Anisometric Poly(ethylene) Glycol Microgels Using a Novel Radical-Free Microfluidic System.

Complex 3D artificial tissue constructs are extensively investigated for tissue regeneration. Frequently, materials and cells are delivered separately without benefitting from the synergistic effect of combined administration. Cell delivery inside a material construct provides the cells with a supportive environment by presenting biochemical, mechanical, and structural signals to direct cell behavior. Conversely, the cell/material interaction is poorly understood at the micron scale and new systems are required to investigate the effect of micron-scale features on cell functionality. Consequently, cells are encapsulated in microgels to avoid diffusion limitations of nutrients and waste and facilitate analysis techniques of single or collective cells. However, up to now, the production of soft cell-loaded microgels by microfluidics is limited to spherical microgels. Here, a novel method is presented to produce monodisperse, anisometric poly(ethylene) glycol microgels to study cells inside an anisometric architecture. These microgels can potentially direct cell growth and can be injected as rod-shaped mini-tissues that further assemble into organized macroscopic and macroporous structures post-injection. Their aspect ratios are adjusted with flow parameters, while mechanical and biochemical properties are altered by modifying the precursors. Encapsulated primary fibroblasts are viable and spread and migrate across the 3D microgel structure.

Solvent-Induced Nanotopographies of Single Microfibers Regulate Cell Mechanotransduction.

The extracellular matrix (ECM) is a dynamic three-dimensional (3D) fibrous network, surrounding all cells in vivo. Fiber manufacturing techniques are employed to mimic the ECM but still lack the knowledge and methodology to produce single fibers approximating cell size with different surface topographies to study cell-material interactions. Using solvent-assisted spinning (SAS), the potential to continuously produce single microscale fibers with unlimited length, precise diameter, and specific surface topographies was demonstrated. By applying solvents with different solubilities and volatilities, fibers with smooth, grooved, and porous surface morphologies are produced. Due to their hierarchical structures, the porous fibers are the most hydrophobic, followed by the grooved and the smooth fibers. The fiber diameter is increased by increasing the polymer concentration or decreasing the collector rotational speed. Moreover, SAS offers the advantage to control the interfiber distance and angle to fabricate multilayered 3D constructs. This report shows for the first time that the micro- and nanoscale topographies of single fibers mechanically regulate cell behavior. Fibroblasts, grown on fibers with grooved topographical features, stretch and elongate more compared to smooth and porous fibers, whereas both porous and grooved fibers induce nuclear translocation of yes-associated protein. The presented technique, therefore, provides a unique platform to study the interaction between cells and single ECM-like fibers in a precise and reproducible manner, which is of great importance for new material developments in the field of tissue engineering.

A catalyst-free, temperature controlled gelation system for in-mold fabrication of microgels.

Anisometric microgels are prepared via thermal crosslinking using an in-mold polymerization technique. Star-shaped poly(ethylene oxide-stat-propylene oxide) polymers, end-modified with amine and epoxy groups, form hydrogels, of which the mechanical properties and gelation rate can be adjusted by the temperature, duration of heating, and polymer concentration. Depending on the microgel stiffness, the rod-shaped microgels self-assemble into ordered or disordered structures.

Biofunctionalized aligned microgels provide 3D cell guidance to mimic complex tissue matrices.

Natural healing is based on highly orchestrated processes, in which the extracellular matrix plays a key role. To resemble the native cell environment, we introduce an artificial extracellular matrix (aECM) with the capability to template hierarchical and anisotropic structures in situ, allowing a minimally-invasive application via injection. Synthetic, magnetically responsive, rod-shaped microgels are locally aligned and fixed by a biocompatible surrounding hydrogel, creating a hybrid anisotropic hydrogel (Anisogel), of which the physical, mechanical, and chemical properties can be tailored. The microgels are rendered cell-adhesive with GRGDS and incorporated either inside a cell-adhesive fibrin or bioinert poly(ethylene glycol) hydrogel to strongly interact with fibroblasts. GRGDS-modified microgels inside a fibrin-based Anisogel enhance fibroblast alignment and lead to a reduction in fibronectin production, indicating successful replacement of structural proteins. In addition, YAP-translocation to the nucleus increases with the concentration of microgels, indicating cellular sensing of the overall anisotropic mechanical properties of the Anisogel. For bioinert surrounding PEG hydrogels, GRGDS-microgels are required to support cell proliferation and fibronectin production. In contrast to fibroblasts, primary nerve growth is not significantly affected by the biomodification of the microgels. In conclusion, this approach opens new opportunities towards advanced and complex aECMs for tissue regeneration.

Hierarchical Design of Tissue Regenerative Constructs.

The worldwide shortage of organs fosters significant advancements in regenerative therapies. Tissue engineering and regeneration aim to supply or repair organs or tissues by combining material scaffolds, biochemical signals, and cells. The greatest challenge entails the creation of a suitable implantable or injectable 3D macroenvironment and microenvironment to allow for ex vivo or in vivo cell-induced tissue formation. This review gives an overview of the essential components of tissue regenerating scaffolds, ranging from the molecular to the macroscopic scale in a hierarchical manner. Further, this review elaborates about recent pivotal technologies, such as photopatterning, electrospinning, 3D bioprinting, or the assembly of micrometer-scale building blocks, which enable the incorporation of local heterogeneities, similar to most native extracellular matrices. These methods are applied to mimic a vast number of different tissues, including cartilage, bone, nerves, muscle, heart, and blood vessels. Despite the tremendous progress that has been made in the last decade, it remains a hurdle to build biomaterial constructs in vitro or in vivo with a native-like structure and architecture, including spatiotemporal control of biofunctional domains and mechanical properties. New chemistries and assembly methods in water will be crucial to develop therapies that are clinically translatable and can evolve into organized and functional tissues.

An Injectable Hybrid Hydrogel with Oriented Short Fibers Induces Unidirectional Growth of Functional Nerve Cells.

To regenerate soft aligned tissues in living organisms, low invasive biomaterials are required to create 3D microenvironments with a structural complexity to mimic the tissue's native architecture. Here, a tunable injectable hydrogel is reported, which allows precise engineering of the construct's anisotropy in situ. This material is defined as an Anisogel, representing a new type of tissue regenerative therapy. The Anisogel comprises a soft hydrogel, surrounding magneto-responsive, cell adhesive, short fibers, which orient in situ in the direction of a low external magnetic field, before complete gelation of the matrix. The magnetic field can be removed after gelation of the biocompatible gel precursor, which fixes the aligned fibers and preserves the anisotropic structure of the Anisogel. Fibroblasts and nerve cells grow and extend unidirectionally within the Anisogels, in comparison to hydrogels without fibers or with randomly oriented fibers. The neurons inside the Anisogel show spontaneous electrical activity with calcium signals propagating along the anisotropy axis of the material. The reported system is simple and elegant and the short magneto-responsive fibers can be produced with an effective high-throughput method, ideal for a minimal invasive route for aligned tissue therapy.

Microfluidic fabrication of polyethylene glycol microgel capsules with tailored properties for the delivery of biomolecules.

Microfluidic encapsulation platforms have great potential not only in pharmaceutical applications but also in the consumer products industry. Droplet-based microfluidics is increasingly used for the production of monodisperse polymer microcapsules for biomedical applications. In this work, a microfluidic technique is developed for the fabrication of monodisperse double emulsion droplets, where the shell is crosslinked into microgel capsules. A six-armed acrylated star-shaped poly(ethylene oxide-stat-propylene oxide) pre-polymer is used to form the microgel shell after a photo-initiated crosslinking reaction. The synthesized microgel capsules are hollow, enabling direct encapsulation of large amounts of multiple biomolecules with the inner aqueous phase completely engulfed inside the double emulsion droplets. The shell thickness and overall microgel sizes can be controlled via the flow rates. The morphology and size of the shells are characterized by cryo-SEM. The encapsulation and retention of 10 kDa FITC-dextran and its microgel degradation mediated release are monitored by fluorescence microscopy.

Nerve Cells Decide to Orient inside an Injectable Hydrogel with Minimal Structural Guidance.

Injectable biomaterials provide the advantage of a minimally invasive application but mostly lack the required structural complexity to regenerate aligned tissues. Here, we report a new class of tissue regenerative materials that can be injected and form an anisotropic matrix with controlled dimensions using rod-shaped, magnetoceptive microgel objects. Microgels are doped with small quantities of superparamagnetic iron oxide nanoparticles (0.0046 vol %), allowing alignment by external magnetic fields in the millitesla order. The microgels are dispersed in a biocompatible gel precursor and after injection and orientation are fixed inside the matrix hydrogel. Regardless of the low volume concentration of the microgels below 3%, at which the geometrical constrain for orientation is still minimum, the generated macroscopic unidirectional orientation is strongly sensed by the cells resulting in parallel nerve extension. This finding opens a new, minimal invasive route for therapy after spinal cord injury.