Interlinked Macroporous 3D Scaffolds from Microgel Rods.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Functionalized Microgel Rods Interlinked into Soft Macroporous Structures for 3D Cell Culture.

In this work, a two component microgel assembly using soft anisometric microgels that interlink to create a 3D macroporous construct for cell growth is reported. Reactive microgel rods with variable aspect ratio are produced via microfluidics in a continuous plug-flow on-chip gelation method by photoinitiated free-radical polymerization of star-polyethylene glycol-acrylate with glycidyl methacrylate or 2-aminoethyl methacrylate comonomers. The resulting complementary epoxy- and amine-functionalized microgels assemble and interlink with each other via a ring opening reaction, resulting in macroporous constructs with pores up to several hundreds of micrometers. The level of crosslinking depends on the functionalization degree of the microgels, which also affects the stiffness and cell adhesiveness of the microgels when modified with the cell-adhesive GRGDS-PC peptide. Therefore, 3D spreading and growth of cells inside the macroporous structure is influenced not only by the presence of macropores but also by the mechanical and biochemical properties of the individual microgels.

Anisometric Microstructures to Determine Minimal Critical Physical Cues Required for Neurite Alignment.

In nerve regeneration, scaffolds play an important role in providing an artificial extracellular matrix with architectural, mechanical, and biochemical cues to bridge the site of injury. Directed nerve growth is a crucial aspect of nerve repair, often introduced by engineered scaffolds imparting linear tracks. The influence of physical cues, determined by well-defined architectures, has been mainly studied for implantable scaffolds and is usually limited to continuous guiding features. In this report, the potential of short anisometric microelements in inducing aligned neurite extension, their dimensions, and the role of vertical and horizontal distances between them, is investigated. This provides crucial information to create efficient injectable 3D materials with discontinuous, in situ magnetically oriented microstructures, like the Anisogel. By designing and fabricating periodic, anisometric, discreet guidance cues in a high-throughput 2D in vitro platform using two-photon lithography techniques, the authors are able to decipher the minimal guidance cues required for directed nerve growth along the major axis of the microelements. These features determine whether axons grow unidirectionally or cross paths via the open spaces between the elements, which is vital for the design of injectable Anisogels for enhanced nerve repair.

Bicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels.

Nerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and 'random screened' tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvß3 or α5ß1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvß3 or α5ß1, compared to monocyclic or linear RGD peptides, with a slight preference to αvß3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration.

Two Robust Strategies toward Hydrogels from Quenched Block Copolymer Nanofibrillar Micelles.

While the formation of (tri)block copolymer hydrogels has been extensively investigated, such studies mostly focused on equilibrium self-assembling whereas the use of preformed structures as building blocks such as out of equilibrium, quenched, nanofibrillar micelles is still a challenge. Here, we demonstrate that quenched, ultralong polystyrene-b-poly(ethylene oxide) (PS-b-PEO) micelles can be used as robust precursors of hydrogels. Two cross-linking strategies, (i) thermal fusion of micellar cores and (ii) chemical cross-linking of preformed micellar coronas were studied. The gelation process and the structure of the micellar networks were investigated by in situ rheological measurements, confocal microscopy and transmission electron microscopy. Direct observation of core fusion of preformed quenched micelles is provided validating this method as a robust gelation route. Using time sweep rheological experiments, it was found for both cross-linking methods that these 3D "mikado" gels are formed in three different stages, containing (1) initiation, (2) transition (growth), and (3) stabilization regimes.

A facile approach for the fabrication of 2D supermicelle networks.

A novel and facile approach to fabricating well-organized macroscopic 2D networks of cylindrical micelles is reported, based on transfer printing and thermal welding of aligned supramolecular micelles of block copolymers. This versatile approach provides a new strategy for fabricating functional 2D superstructures with a higher level of order.