Covalent Grafting of Functionalized MEW Fibers to Silk Fibroin Hydrogels to Obtain Reinforced Tissue Engineered Constructs.

Hydrogels are ideal materials to encapsulate cells, making them suitable for applications in tissue engineering and regenerative medicine. However, they generally do not possess adequate mechanical strength to functionally replace human tissues, and therefore they often need to be combined with reinforcing structures. While the interaction at the interface between the hydrogel and reinforcing structure is imperative for mechanical function and subsequent biological performance, this interaction is often overlooked. Melt electrowriting enables the production of reinforcing microscale fibers that can be effectively integrated with hydrogels. Yet, studies on the interaction between these micrometer scale fibers and hydrogels are limited. Here, we explored the influence of covalent interfacial interactions between reinforcing structures and silk fibroin methacryloyl hydrogels (silkMA) on the mechanical properties of the construct and cartilage-specific matrix production in vitro. For this, melt electrowritten fibers of a thermoplastic polymer blend (poly(hydroxymethylglycolide-co-ε-caprolactone):poly(ε-caprolactone) (pHMGCL:PCL)) were compared to those of the respective methacrylated polymer blend pMHMGCL:PCL as reinforcing structures. Photopolymerization of the methacrylate groups, present in both silkMA and pMHMGCL, was used to generate hybrid materials. Covalent bonding between the pMHMGCL:PCL blend and silkMA hydrogels resulted in an elastic response to the application of torque. In addition, an improved resistance was observed to compression (∼3-fold) and traction (∼40-55%) by the scaffolds with covalent links at the interface compared to those without these interactions. Biologically, both types of scaffolds (pHMGCL:PCL and pMHMGCL:PCL) showed similar levels of viability and metabolic activity, also compared to frequently used PCL. Moreover, articular cartilage progenitor cells embedded within the reinforced silkMA hydrogel were able to form a cartilage-like matrix after 28 days of in vitro culture. This study shows that hybrid cartilage constructs can be engineered with tunable mechanical properties by grafting silkMA hydrogels covalently to pMHMGCL:PCL blend microfibers at the interface.

Hyaluronic Acid-PEG-Based Diels-Alder In Situ Forming Hydrogels for Sustained Intraocular Delivery of Bevacizumab.

Retinal diseases are the leading cause of visual impairment worldwide. The effectiveness of antibodies for the treatment of retinal diseases has been demonstrated. Despite the clinical success, achieving sufficiently high concentrations of these protein therapeutics at the target tissue for an extended period is challenging. Patients suffering from macular degeneration often receive injections once per month. Therefore, there is a growing need for suitable systems that can help reduce the number of injections and adverse effects while improving patient complacency. This study systematically characterized degradable "in situ" forming hydrogels that can be easily injected into the vitreous cavity using a small needle (29G). After intravitreal injection, the formulation is designed to undergo a sol-gel phase transition at the administration site to obtain an intraocular depot system for long-term sustained release of bioactives. A Diels-Alder reaction was exploited to crosslink hyaluronic acid-bearing furan groups (HAFU) with 4 arm-PEG10K-maleimide (4APM), yielding stable hydrogels. Here, a systematic investigation of the effects of polymer composition and the ratio between functional groups on the physicochemical properties of hydrogels was performed to select the most suitable formulation for protein delivery. Rheological analysis showed rapid hydrogel formation, with the fastest gel formation within 5 min after mixing the hydrogel precursors. In this study, the mechanical properties of an ex vivo intravitreally formed hydrogel were investigated and compared to the in vitro fabricated samples. Swelling and degradation studies showed that the hydrogels are biodegradable by the retro-Diels-Alder reaction under physiological conditions. The 4APM-HAFU (ratio 1:5) hydrogel formulation showed sustained release of bevacizumab > 400 days by a combination of diffusion, swelling, and degradation. A bioassay showed that the released bevacizumab remained bioactive. The hydrogel platform described in this study offers high potential for the sustained release of therapeutic antibodies to treat ocular diseases.

Viscoelastic Chondroitin Sulfate and Hyaluronic Acid Double-Network Hydrogels with Reversible Cross-Links.

Viscoelastic hydrogels are gaining interest as they possess necessary requirements for bioprinting and injectability. By means of reversible, dynamic covalent bonds, it is possible to achieve features that recapitulate the dynamic character of the extracellular matrix. Dually cross-linked and double-network (DN) hydrogels seem to be ideal for the design of novel biomaterials and bioinks, as a wide range of properties required for mimicking advanced and complex tissues can be achieved. In this study, we investigated the fabrication of chondroitin sulfate/hyaluronic acid (CS/HA)-based DN hydrogels, in which two networks are interpenetrated and cross-linked with the dynamic covalent bonds of very different lifetimes. Namely, Diels-Alder adducts (between methylfuran and maleimide) and hydrazone bonds (between aldehyde and hydrazide) were chosen as cross-links, leading to viscoelastic hydrogels. Furthermore, we show that viscoelasticity and the dynamic character of the resulting hydrogels could be tuned by changing the composition, that is, the ratio between the two types of cross-links. Also, due to a very dynamic nature and short lifetime of hydrazone cross-links (∼800 s), the DN hydrogel is easily processable (e.g., injectable) in the first stages of gelation, allowing the material to be used in extrusion-based 3D printing. The more long-lasting and robust Diels-Alder cross-links are responsible for giving the network enhanced mechanical strength and structural stability. Being highly charged and hydrophilic, the cross-linked CS and HA enable a high swelling capacity (maximum swelling ratio ranging from 6 to 12), which upon confinement results in osmotically stiffened constructs, able to mimic the mechanical properties of cartilage tissue, with the equilibrium moduli ranging from 0.3 to 0.5 MPa. Moreover, the mesenchymal stromal cells were viable in the presence of the hydrogels, and the effect of the degradation products on the macrophages suggests their safe use for further translational applications. The DN hydrogels with dynamic covalent cross-links hold great potential for the development of novel smart and tunable viscoelastic materials to be used as biomaterial inks or bioinks in bioprinting and regenerative medicine.

A Chemically Defined Hydrogel for Human Liver Organoid Culture.

End-stage liver diseases are an increasing health burden, and liver transplantations are currently the only curative treatment option. Due to a lack of donor livers, alternative treatments are urgently needed. Human liver organoids are very promising for regenerative medicine; however, organoids are currently cultured in Matrigel, which is extracted from the extracellular matrix of the Engelbreth-Holm-Swarm mouse sarcoma. Matrigel is poorly defined, suffers from high batch-to-batch variability and is of xenogeneic origin, which limits the clinical application of organoids. Here, a novel hydrogel based on polyisocyanopeptides (PIC) and laminin-111 is described for human liver organoid cultures. PIC is a synthetic polymer that can form a hydrogel with thermosensitive properties, making it easy to handle and very attractive for clinical applications. Organoids in an optimized PIC hydrogel proliferate at rates comparable to those observed with Matrigel; proliferation rates are stiffness-dependent, with lower stiffnesses being optimal for organoid proliferation. Moreover, organoids can be efficiently differentiated toward a hepatocyte-like phenotype with key liver functions. This proliferation and differentiation potential maintain over at least 14 passages. The results indicate that PIC is very promising for human liver organoid culture and has the potential to be used in a variety of clinical applications including cell therapy and tissue engineering.

Microstructured silk fiber scaffolds with enhanced stretchability.

Despite extensive research, current methods for creating three-dimensional (3D) silk fibroin (SF) scaffolds lack control over molecular rearrangement, particularly in the formation of ß-sheet nanocrystals that severely embrittle SF, as well as hierarchical fiber organization at both micro- and macroscale. Here, we introduce a fabrication process based on electrowriting of aqueous SF solutions followed by post-processing using an aqueous solution of sodium dihydrogen phosphate (NaH2PO4). This approach enables gelation of SF chains via controlled ß-sheet formation and partial conservation of compliant random coil structures. Moreover, this process allows for precise architecture control in microfiber scaffolds, enabling the creation of 3D flat and tubular macro-geometries with square-based and crosshatch microarchitectures, featuring inter-fiber distances of 400 µm and ∼97% open porosity. Remarkably, the crosslinked printed structures demonstrated a balanced coexistence of ß-sheet and random coil conformations, which is uncommon for organic solvent-based crosslinking methods. This synergy of printing and post-processing yielded stable scaffolds with high compliance (modulus = 0.5-15 MPa) and the ability to support elastic cyclic loading up to 20% deformation. Furthermore, the printed constructs supported in vitro adherence and growth of human renal epithelial and endothelial cells with viability above 95%. These cells formed homogeneous monolayers that aligned with the fiber direction and deposited type-IV collagen as a specific marker of healthy extracellular matrix, indicating that both cell types attach, proliferate, and organize their own microenvironment within the SF scaffolds. These findings represent a significant development in fabricating organized stable SF scaffolds with unique microfiber structures and mechanical and biological properties that make them highly promising for tissue engineering applications.

3D culture of ovarian follicles in granular and nanofibrillar hydrogels.

3D culture of ovarian follicles in hydrogel matrices is an important emerging tool for basic scientific studies as well as clinical applications such as fertility preservation. For optimizing and scaling 3D culture of preantral follicles, there is a need for identifying biomaterial matrices that simplifies and improves the current culture procedures. At present, microencapsulation of follicles in alginate beads is the most commonly used approach. However, this technique involves notable manual handling and is best suited for encapsulation of single or several follicles. As a potential alternative, we here explore the suitability of different particle-based hydrogel matrices, where follicles can easily be introduced in tunable 3D environments, in large numbers. Specifically, we study the growth of secondary murine follicles in microgranular alginate and nanofibrillar cellulose matrices, with and without cell-binding cues, and map follicle growth against the viscoelastic properties of the matrices. We cultured follicles within the particle-based hydrogels for 10 days and continuously monitored their size, survival, and tendency to extrude oocytes. Interestingly, we observed that the diameter of the growing follicles increased significantly in the particle-based matrices, as compared to state-of-the-art alginate micro-encapsulation. On the other hand, the follicles displayed an increased tendency for early oocyte extrusion in the granular matrices, leading to a notable reduction in the number of intact follicles. We propose that this may be caused by impaired diffusion of nutrients and oxygen through thicker matrices, attributable to our experimental setup. Still, our findings suggest that viscoelastic, granular hydrogels represent promising matrices for 3D culture of early-stage ovarian follicles. In particular, these materials may easily be implemented in advanced culturing devices such as micro-perfusion systems.

Transparent and Cell-Guiding Cellulose Nanofiber 3D Printing Bioinks.

For three-dimensional (3D) bioprinting to fulfill its promise and enable the automated fabrication of complex tissue-mimicking constructs, there is a need for developing bioinks that are not only printable and biocompatible but also have integrated cell-instructive properties. Toward this goal, we here present a scalable technique for generating nanofiber 3D printing inks with unique tissue-guiding capabilities. Our core methodology relies on tailoring the size and dispersibility of cellulose fibrils through a solvent-controlled partial carboxymethylation. This way, we generate partially negatively charged cellulose nanofibers with diameters of ∼250 nm and lengths spanning tens to hundreds of microns. In this range, the fibers structurally match the size and dimensions of natural collagen fibers making them sufficiently large to orient cells. Yet, they are simultaneously sufficiently thin to be optically transparent. By adjusting fiber concentration, 3D printing inks with excellent shear-thinning properties can be established. In addition, as the fibers are readily dispersible, composite inks with both carbohydrates and extracellular matrix (ECM)-derived proteins can easily be generated. We apply such composite inks for 3D printing cell-laden and cross-linkable structures, as well as tissue-guiding gel substrates. Interestingly, we find that the spatial organization of engineered tissues can be defined by the shear-induced alignment of fibers during the printing procedure. Specifically, we show how myotubes derived from human and murine skeletal myoblasts can be programmed into linear and complex nonlinear architectures on soft printed substrates with intermediate fiber contents. Our nanofibrillated cellulose inks can thus serve as a simple and scalable tool for engineering anisotropic human muscle tissues that mimic native structure and function.

Local changes in microtubule network mobility instruct neuronal polarization and axon specification.

The polarization of neurons into axons and dendrites depends on extracellular cues, intracellular signaling, cytoskeletal rearrangements, and polarized transport, but the interplay between these processes during polarization remains unresolved. Here, we show that axon specification is determined by differences in microtubule network mobility between neurites, regulated by Rho guanosine triphosphatases (GTPases) and extracellular cues. In developing neurons, retrograde microtubule flow prevents the entry of the axon-selective motor protein Kinesin-1 into most neurites. Using inducible assays to control microtubule network flow, we demonstrate that local inhibition of microtubule mobility is sufficient to guide Kinesin-1 into a specific neurite, whereas long-term global inhibition induces the formation of multiple axons. We furthermore show that extracellular mechanical cues and intracellular Rho GTPase signaling control the local differences in microtubule network flow. These results reveal a novel cytoskeletal mechanism for neuronal polarization.

Creating a Functional Biomimetic Cartilage Implant Using Hydrogels Based on Methacrylated Chondroitin Sulfate and Hyaluronic Acid.

The load-bearing function of articular cartilage tissue contrasts with the poor load-bearing capacity of most soft hydrogels used for its regeneration. The present study explores whether a hydrogel based on the methacrylated natural polymers chondroitin sulfate (CSMA) and hyaluronic acid (HAMA), injected into warp-knitted spacer fabrics, could be used to create a biomimetic construct with cartilage-like mechanical properties. The swelling ratio of the combined CSMA/HAMA hydrogels in the first 20 days was higher for hydrogels with a higher CSMA concentration, and these hydrogels also degraded quicker, whereas those with a 1.33 wt% of HAMA were stable for more than 120 days. When confined by a polyamide 6 (PA6) spacer fabric, the volumetric swelling of the combined CSMA/HAMA gels (10 wt%, 6.5 × CSMA:HAMA ratio) was reduced by ~53%. Both the apparent peak and the equilibrium modulus significantly increased in the PA6-restricted constructs compared to the free-swelling hydrogels after 28 days of swelling, and no significant differences in the moduli and time constant compared to native bovine cartilage were observed. Moreover, the cell viability in the CSMA/HAMA PA6 constructs was comparable to that in gelatin-methacrylamide (GelMA) PA6 constructs at one day after polymerization. These results suggest that using a HydroSpacer construct with an extracellular matrix (ECM)-like biopolymer-based hydrogel is a promising approach for mimicking the load-bearing properties of native cartilage.

Hyaluronic acid and chondroitin sulfate (meth)acrylate-based hydrogels for tissue engineering: Synthesis, characteristics and pre-clinical evaluation.

Hydrogels based on photocrosslinkable Hyaluronic Acid Methacrylate (HAMA) and Chondroitin Sulfate Methacrylate (CSMA) are presently under investigation for tissue engineering applications. HAMA and CSMA gels offer tunable characteristics such as tailorable mechanical properties, swelling characteristics, and enzymatic degradability. This review gives an overview of the scientific literature published regarding the pre-clinical development of covalently crosslinked hydrogels that (partially) are based on HAMA and/or CSMA. Throughout the review, recommendations for the next steps in clinical translation of hydrogels based on HAMA or CSMA are made and potential pitfalls are defined. Specifically, a myriad of different synthetic routes to obtain polymerizable hyaluronic acid and chondroitin sulfate derivatives are described. The effects of important parameters such as degree of (meth)acrylation and molecular weight of the synthesized polymers on the formed hydrogels are discussed and useful analytical techniques for their characterization are summarized. Furthermore, the characteristics of the formed hydrogels including their enzymatic degradability are discussed. Finally, a summary of several recent applications of these hydrogels in applied fields such as cartilage and cardiac regeneration and advanced tissue modelling is presented.

Carbon Nanotube Reinforced Supramolecular Hydrogels for Bioapplications.

Nanocomposite hydrogels based on carbon nanotubes (CNTs) are known to possess remarkable stiffness, electrical, and thermal conductivity. However, they often make use of CNTs as fillers in covalently cross-linked hydrogel networks or involve direct cross-linking between CNTs and polymer chains, limiting processability properties. Herein, nanocomposite hydrogels are developed, in which CNTs are fillers in a physically cross-linked hydrogel. Supramolecular nanocomposites are prepared at various CNT concentrations, ranging from 0.5 to 6 wt%. Incorporation of 3 wt% of CNTs leads to an increase of the material's toughness by over 80%, and it enhances electrical conductivity by 358%, compared to CNT-free hydrogel. Meanwhile, the nanocomposite hydrogels maintain thixotropy and processability, typical of the parent hydrogel. The study also demonstrates that these materials display remarkable cytocompatibility and support cell growth and proliferation, while preserving their functional activities. These supramolecular nanocomposite hydrogels are therefore promising candidates for biomedical applications, in which both toughness and electrical conductivity are important parameters.

Effects of Surfactant and Urea on Dynamics and Viscoelastic Properties of Hydrophobically Assembled Supramolecular Hydrogel.

Physically associated hydrogels based on strong hydrophobic interactions often have attractive mechanical properties that combine processability with elasticity. However, there is a need to study such interactions and understand their relation to the macroscopic hydrogel properties. Therefore, we use the surfactant sodium dodecyl sulfate (SDS) and urea as reagents that disrupt hydrophobic interactions. The model hydrogel is based on a segmented copolymer between poly(ethylene glycol) (PEG) and hydrophobic dimer fatty acid (DFA). We show that both agents influence viscoelastic properties, dynamics, and relaxation processes of the model hydrogel. In particular, the relaxation time is significantly reduced by urea, as compared to SDS, whereas the surfactant causes a decrease of the modulus of the hydrogel more efficiently. The reversibility of the effects of SDS and urea can be exploited, for instance, by using an injectable sol that solidifies when the SDS or urea diffuses out of the sample. Surfactant-induced processability may be advantageous in future applications of hydrophobically assembled physical hydrogels.

Tough Supramolecular Hydrogel Based on Strong Hydrophobic Interactions in a Multiblock Segmented Copolymer.

We report the preparation and structural and mechanical characterization of a tough supramolecular hydrogel, based exclusively on hydrophobic association. The system consists of a multiblock, segmented copolymer of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic dimer fatty acid (DFA) building blocks. A series of copolymers containing 2K, 4K, and 8K PEG were prepared. Upon swelling in water, a network is formed by self-assembly of hydrophobic DFA units in micellar domains, which act as stable physical cross-link points. The resulting hydrogels are noneroding and contain 75-92 wt % of water at swelling equilibrium. Small-angle neutron scattering (SANS) measurements showed that the aggregation number of micelles ranges from 2 × 102 to 6 × 102 DFA units, increasing with PEG molecular weight. Mechanical characterization indicated that the hydrogel containing PEG 2000 is mechanically very stable and tough, possessing a tensile toughness of 4.12 MJ/m3. The high toughness, processability, and ease of preparation make these hydrogels very attractive for applications where mechanical stability and load bearing features of soft materials are required.