Poly(2‐oxazoline) hydrogels crosslinked with aliphatic bis(2‐oxazoline)s: Properties,cytotoxicity, and cell cultivation

A series of hydrogels from 2‐ethyl‐2‐oxazoline and three bis(2‐oxazoline) crosslinkers—1,4‐butylene‐2,2′‐bis(2‐oxazoline), 1,6‐hexamethylene‐2,2′‐bis(2‐oxazoline), and 1,8‐octamethylene‐2,2′‐bis(2‐oxazoline)—are prepared. The hydrogels differ by the length of aliphatic chain of crosslinker and by the percentage of crosslinker (2–10%). The influence of the type and the percentage of the crosslinker on swelling properties, mechanical properties, and state of water is studied. The equilibrium swelling degree in water ranges from 2 to 20. With a proper selection of the crosslinker, Young's modulus can be varied from 10 kPa to almost 100 kPa. To evaluate the potential for medical applications, the cytotoxicity of extracts and the contact toxicity toward murine fibroblasts are measured. The hydrogels with the crosslinker containing a shorter aliphatic exhibit low toxicity toward fibroblast cells. Moreover, the viability and the proliferation of pancreatic β‐cells incubated inside hydrogels for 12 days are analyzed. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1548–1559     

Tunable Pentapeptide Self‐Assembled β‐Sheet Hydrogels

Oligopeptide‐based supramolecular hydrogels hold promise in a range of applications. The gelation of these systems is hard to control, with minor alterations in the peptide sequence significantly influencing the self‐assembly process. We explored three pentapeptide sequences with different charge distributions and discovered that they formed robust, pH‐responsive hydrogels. By altering the concentration and charge distribution of the peptide sequence, the stiffness of the hydrogels could be tuned across two orders of magnitude (2–200 kPa). Also, through reassembly of the β‐sheet interactions the hydrogels could self‐heal and they demonstrated shear‐thin behavior. Using spectroscopic and cryo‐imaging techniques, we investigated the relationship between peptide sequence and molecular structure, and how these influence the mechanical properties of the hydrogel. These pentapeptide hydrogels with tunable morphology and mechanical properties have promise in tissue engineering, injectable delivery vectors, and 3D printing applications.     

Drug‐Loaded Elastin‐Like Polypeptide–Collagen Hydrogels with High Modulus for Bone Tissue Engineering

Emphasizing the role of hydrogel stiffness and cellular differentiation, this study develops collagen and elastin‐like polypeptide (ELP)–based bone regenerative hydrogels loaded with recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and doxycycline with mechanical properties suitable for osteogenesis. The drug‐incorporated collagen–ELP hydrogels has significantly higher modulus of 35 ± 5 kPa compared to collagen‐only hydrogels. Doxycycline shows a bi‐phasic release with an initial burst release followed by a gradual release, while rhBMP‐2 exhibits a nearly linear release profile for all hydrogels. The released doxycycline shows anti‐microbial activity against Pseudomonas aeruginosa, Streptococcus sanguinis, and Escherichia coli. Microscopic observation of the hydrogels reveals their interconnected, macroporous, 3D open architecture with pore diameters between 160 and 400 µm. This architecture supports human adipose–derived stem cell attachment and proliferation from initial days of cell seeding, forming a thick cellular sheath by day 21. Interestingly, in collagen and collagen–ELP hydrogels, cell morphology is elongated with stretched slender lamellipodial formation, while cells assemble as spheroidal aggregates in crosslinked as well as drug‐loaded hydrogels. Osteogenic markers, alkaline phosphatase and osteocalcin, are expressed maximally for drug‐loaded hydrogels compared to those without drugs. The drug‐loaded collagen–ELP hydrogels are thus promising for combating bacterial infection and promoting guided bone regeneration.     

pH‐Sensitive Mechanical Properties of Elastin‐Based Hydrogels

Ionizable amino acids in protein‐based hydrogels can confer pH‐responsive behavior. Because elastin‐like polypeptides (ELPs) have an established sequence and can crosslink to form hydrogels, they are an ideal system for creating pH‐sensitive materials. This study examines different parameters that might affect pH‐sensitive behavior and characterizes the mechanical and physical properties between pH 3 and 11 of three ELP‐based crosslinked hydrogels. The first finding is that varying the amount of crosslinker affects the overall stiffness and resilience of the hydrogels but does not strongly affect water content, swelling ratio, or pH sensitivity. Second, the choice of two popular tag sequences, which vary in histidine and aspartic acid content, does not have a strong effect on pH‐sensitive properties. Last, selectively blocking lysine and tyrosine residues through acetylation significantly decreases the pH‐sensitive zeta potential. Acetylated hydrogels also demonstrate different behavior at low pH values with reduced swelling, reduced water content, and higher stiffness. Overall, this work demonstrates that ELP hydrogels with ionizable groups are promising materials for environmentally‐responsive applications such as drug delivery, tissue engineering, and microfluidics.     

Redox‐Responsive Resilin‐Like Hydrogels for Tissue Engineering and Drug Delivery Applications

Resilin, a protein found in insect cuticles, is renowned for its outstanding elastomeric properties. The authors' laboratory previously developed a recombinant protein, which consisted of consensus resilin‐like repeats from Anopheles gambiae, and demonstrated its potential in cartilage and vascular engineering. To broaden the versatility of the resilin‐like protein, this study utilizes a cleavable crosslinker, which contains a disulfide bond, to develop smart resilin‐like hydrogels that are redox‐responsive. The hydrogels exhibit a porous structure and a stable storage modulus (G′) of ≈3 kPa. NIH/3T3 fibroblasts cultured on hydrogels for 24 h have a high viability (>95%). In addition, the redox‐responsive hydrogels show significant degradation in a reducing environment (10 mm glutathione (GSH)). The release profiles of fluorescently labeled dextrans encapsulated within the hydrogels are assessed in vitro. For dextran that is estimated to be larger than the mesh size of the gel, faster release is observed in the presence of reducing agents due to degradation of the hydrogel networks. These studies thus demonstrate the potential of using these smart hydrogels in a variety of applications ranging from scaffolds for tissue engineering to drug delivery systems that target the intracellular reductive environments of tumors.     

Chondrocyte Generation of Cartilage‐Like Tissue Following Photoencapsulation in Methacrylated Polysaccharide Solution Blends

Chondrocyte‐seeded, photo‐cross‐linked hydrogels prepared from solutions containing 50% mass fractions of methacrylated glycol chitosan or methacrylated hyaluronic acid (MHA) with methacrylated chondroitin sulfate (MCS) are cultured in vitro under static conditions over 35 d to assess their suitability for load‐bearing soft tissue repair. The photo‐cross‐linked hydrogels have initial equilibrium moduli between 100 and 300 kPa, but only the MHAMCS hydrogels retain an approximately constant modulus (264 ± 5 kPa) throughout the culture period. Visually, the seeded chondrocytes in the MHAMCS hydrogels are well distributed with an apparent constant viability in culture. Multicellular aggregates are surrounded by cartilaginous matrix, which contain aggrecan and collagen II. Thus, co‐cross‐linked MCS and MHA hydrogels may be suited for use in an articular cartilage or nucleus pulposus repair applications.

     

Enhanced Cellular Activity in Gelatin‐Poly(Ethylene Glycol) Hydrogels without Compromising Gel Stiffness

Adhesion and proliferation of cells are often suppressed in rigid hydrogels as gel stiffness induces mechanical stress to embedded cells. Herein, the composite hydrogel systems to facilitate high cellular activities are described, while maintaining relatively high gel stiffness. This unusual property is obtained by harmonizing gelatin‐poly(ethylene glycol)‐tyramine (GPT, semisynthetic polymer) and gelatin‐hydroxyphenyl propionic acid conjugates (GH, natural polymer) into hydrogels. A minimum GH concentration of 50% is necessary for cells to be proliferative. GPT is utilized to improve biological stability (>1 week) and gelation time (<20 s) of the hydrogels. These results suggest that deficiency in cellular activity driven by gel stiffness could be overcome by finely tuning the material properties in the microenvironments.

     

Non‐Newtonian Polymer–Nanoparticle Hydrogels Enhance Cell Viability during Injection

Drug delivery and cell transplantation require minimally invasive deployment strategies such as injection through clinically relevant high‐gauge needles. Supramolecular hydrogels comprising dodecyl‐modified hydroxypropylmethylcellulose and poly(ethylene glycol)‐block‐poly(lactic acid) have been previously demonstrated for the delivery of drugs and proteins. Here, it is demonstrated that the rheological properties of these hydrogels allow for facile injectability, an increase of cell viability after injection when compared to cell viabilities of cells injected in phosphate‐buffered saline, and homogeneous cell suspensions that do not settle. These hydrogels are injected at 1 mL min^?1 with pressures less than 400 kPa, despite the solid‐like properties of the gel when at rest. The cell viabilities immediately after injection are greater than 86% for adult human dermal fibroblasts, human umbilical vein cells, smooth muscle cells, and human mesenchymal stem cells. Cells are shown to remain suspended and proliferate in the hydrogel at the same rate as observed in cell media. The work expands on the versatility of these hydrogels and lays a foundation for the codelivery of drugs, proteins, and cells.     

Stimuli‐Responsive Biomolecule‐Based Hydrogels and Their Applications

This Review presents polysaccharides, oligosaccharides, nucleic acids, peptides, and proteins as functional stimuli‐responsive polymer scaffolds that yield hydrogels with controlled stiffness. Different physical or chemical triggers can be used to structurally reconfigure the crosslinking units and control the stiffness of the hydrogels. The integration of stimuli‐responsive supramolecular complexes and stimuli‐responsive biomolecular units as crosslinkers leads to hybrid hydrogels undergoing reversible triggered transitions across different stiffness states. Different applications of stimuli‐responsive biomolecule‐based hydrogels are discussed. The assembly of stimuli‐responsive biomolecule‐based hydrogel films on surfaces and their applications are discussed. The coating of drug‐loaded nanoparticles with stimuli‐responsive hydrogels for controlled drug release is also presented.     

Self‐assembled graphene oxide–gelatin nanocomposite hydrogels: Characterization,formation mechanisms,and pH‐sensitive drug release behavior

Novel physically crosslinked graphene oxide (GO)‐gelatin nanocomposite hydrogels were obtained by self‐assembly. The hydrogels with various ratios of GO to gelatin were prepared, and characterized by X‐ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, and scanning electron microscopy. The static and dynamic rheological properties of the hydrogels were investigated, along with the underlying hydrogel formation mechanisms. The storage modulus of the hydrogels (containing 98–98.5 wt % water) reached 114.5 kPa, owing to the relatively strong physical bonding (i.e., hydrogen bonding and electrostatic forces) between GO and gelatin. Drug release tests showed that the drug release from the hydrogel was pH‐dependent, with 96% of the model drug released in a neutral environment, compared to 28% released in an acidic medium. These hydrogels could have potential in pH‐sensitive drug delivery. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 356–367     

Bioactive Phenylboronic Acid-Functionalized Hyaluronic Acid Hydrogels Induce Chondro-Aggregates and Promote Chondrocyte Phenotype

Hydrogels are extensively investigated as biomimetic extracellular matrix (ECM) scaffolds in tissue engineering. The physiological properties of ECM affect cellular behaviors, which is an inspiration for cell-based therapies. Photocurable hyaluronic acid (HA) hydrogel (AHAMA-PBA) modified with 3-aminophenylboronic acid, sodium periodate, and methacrylic anhydride simultaneously is constructed in this study. Chondrocytes are then cultured on the surface of the hydrogels to evaluate the effect of the physicochemical properties of the hydrogels on modulating cellular behaviors. Cell viability assays demonstrate that the hydrogel is non-toxic to chondrocytes. The existence of phenylboronic acid (PBA) moieties enhances the interaction of chondrocytes and hydrogel, promoting cell adhesion and aggregation through filopodia. RT-PCR indicates that the gene expression levels of type II collagen, Aggrecan, and Sox9 are significantly up-regulated in chondrocytes cultured on hydrogels. Moreover, the mechanical properties of the hydrogels have a significant effect on the cell phenotype, with soft gels (≈2 kPa) promoting chondrocytes to exhibit a hyaline phenotype. Overall, PBA-functionalized HA hydrogel with low stiffness exhibits the best effect on promoting the chondrocyte phenotype, which is a promising biomaterial for cartilage regeneration.     

Photo‐crosslinkable hydrogel‐based 3D microfluidic culture device

We developed the photo‐crosslinkable hydrogel‐based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo‐crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular‐shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel‐based 3D microfluidic device, showing that 53–75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo‐crosslinkable hydrogel‐based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.     

Designing Visible Light‐Cured Thiol‐Acrylate Hydrogels for Studying the HIPPO Pathway Activation in Hepatocellular Carcinoma Cells

Various polymerization mechanisms have been developed to prepare peptide‐immobilized poly(ethylene glycol) (PEG) hydrogels, a class of biomaterials suitable for studying cell biology in vitro. Here, a visible light mediated thiol‐acrylate photopolymerization scheme is reported to synthesize dually degradable PEG‐peptide hydrogels with controllable crosslinking and degradability. The influence of immobilized monothiol pendant peptide is systematically evaluated on the crosslinking of these hydrogels. Further, methods are proposed to modulate hydrogel crosslinking, including adjusting concentration of comonomer or altering the design of multifunctional peptide crosslinker. Due to the formation of thioether ester bonds, these hydrogels are hydrolytically degradable. If the dithiol peptide linkers used are susceptible to protease cleavage, these thiol‐acrylate hydrogels can be designed to undergo partial proteolysis. The differences between linear and multiarm PEG‐acrylate (i.e., PEGDA vs PEG4A) are also evaluated. Finally, the use of the mixed‐mode thiol‐acrylate PEG4A‐peptide hydrogels is explored for in situ encapsulation of hepatocellular carcinoma cells (Huh7). The effects of matrix stiffness and integrin binding motif (e.g., RGDS) on Huh7 cell growth and HIPPO pathway activation are studied using PEG4A‐peptide hydrogels. This visible light poly­merized thiol‐acrylate hydrogel system represents an alternative to existing light‐cured hydrogel platforms and shall be useful in many biomedical applications.     

Hydrogels with Reversible Mechanics to Probe Dynamic Cell Microenvironments

The relationship between ECM mechanics and cell behavior is dynamic, as cells remodel and respond to changes in their local environment. Most in vitro substrates are static and supraphysiologically stiff; thus, platforms with dynamic and reversible mechanical changes are needed. Herein, we developed hyaluronic acid‐based substrates capable of sequential photodegradation and photoinitiated crosslinking reactions to soften and then stiffen the hydrogels over a physiologically relevant range of moduli. Reversible mechanical signaling to adhered cells was demonstrated with human mesenchymal stem cells. In situ hydrogel softening (from ca. 14 to 3.5 kPa) led to a decrease in the cell area and nuclear localization of YAP/TAZ, and subsequent stiffening (from ca. 3.5 to 28 kPa) increased the cell area and nuclear localization of YAP/TAZ. Each photoreaction was cytocompatible and tunable, rendering this platform amenable to studies of dynamic mechanics on cell behavior across many cell types and contexts.     

Light-Based 3D Printing of Gelatin-Based Biomaterial Inks to Create a Physiologically Relevant In Vitro Fish Intestinal Model

To provide prominent accessibility of fishmeal to the European population, the currently available, time- and cost-extensive feeding trials, which evaluate fish feed, should be replaced. The current paper reports on the development of a novel 3D culture platform, mimicking the microenvironment of the intestinal mucosa in vitro. The key requirements of the model include sufficient permeability for nutrients and medium-size marker molecules (equilibrium within 24 h), suitable mechanical properties (G' < 10 kPa), and close morphological similarity to the intestinal architecture. To enable processability with light-based 3D printing, a gelatin-methacryloyl-aminoethyl-methacrylate-based biomaterial ink is developed and combined with Tween 20 as porogen to ensure sufficient permeability. To assess the permeability properties of the hydrogels, a static diffusion setup is utilized, indicating that the hydrogel constructs are permeable for a medium size marker molecule (FITC-dextran 4 kg mol⁻¹). Moreover, the mechanical evaluation through rheology evidence a physiologically relevant scaffold stiffness (G' = 4.83 ± 0.78 kPa). Digital light processing-based 3D printing of porogen-containing hydrogels results in the creation of constructs exhibiting a physiologically relevant microarchitecture as evidenced through cryo-scanning electron microscopy. Finally, the combination of the scaffolds with a novel rainbow trout (Oncorhynchus mykiss) intestinal epithelial cell line (RTdi-MI) evidence scaffold biocompatibility.     

Dynamic mechanical properties and swelling of UV‐photopolymerized anionic hydrogels

The tensile dynamic mechanical properties and weight degree of swelling for anionic 2‐hydroxyethyl methacrylate‐co‐acrylic acid hydrogels were observed. Fabrication parameters examined were UV‐photopolymerization exposure time, UV‐photopolymerization intensity, and weight percentage crosslinker. The environmental conditions tested were electrolyte compositions of 0.5 and 0.05 M potassium hydroxide under applied frequencies of 0.1, 1, or 10 Hz. The overall maximum and minimum storage modulus was 1.83 ± 0.18 MPa and 68.5 ± 7.2 kPa, respectively, loss modulus was 432 ± 63 and 7.67 ± 3.22 kPa, respectively, and weight degree of swelling was 14.27 ± 1.27 and 1.95 ± 0.33, respectively. The morphology of fabricated hydrogels was examined using scanning electron microscopy showing a range of porous structures over the fabrication and environmental conditions examined, accounting for the variation in mechanical properties. The properties examined are of interest to researchers fabricating, designing, or modeling active hydrogel‐based microfluidic components. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012     

Cellular Response to Reagent‐Free Electron‐Irradiated Gelatin Hydrogels

As a biomaterial, it is well established that gelatin exhibits low cytotoxicity and can promote cellular growth. However, to circumvent the potential toxicity of chemical crosslinkers, reagent‐free crosslinking methods such as electron irradiation are highly desirable. While high energy irradiation has been shown to exhibit precise control over the degree of crosslinking, these hydrogels have not been thoroughly investigated for biocompatibility and degradability. Here, NIH 3T3 murine fibroblasts are seeded onto irradiated gelatin hydrogels to examine the hydrogel's influence on cellular viability and morphology. The average projected area of cells seeded onto the hydrogels increases with irradiation dose, which correlates with an increase in the hydrogel's shear modulus up to 10 kPa. Cells on these hydrogels are highly viable and exhibits normal cell cycles, particularly when compared to those grown on glutaraldehyde crosslinked gelatin hydrogels. However, proliferation is reduced on both types of crosslinked samples. To mimic the response of the hydrogels in physiological conditions, degradability is monitored in simulated body fluid to reveal strongly dose‐dependent degradation times. Overall, given the low cytotoxicity, influence on cellular morphology and variability in degradation times of the electron irradiated gelatin hydrogels, there is significant potential for application in areas ranging from regenerative medicine to mechanobiology.

     

Fabrication of Stiffness Gradients of GelMA Hydrogels Using a 3D Printed Micromixer

Many properties in both healthy and pathological tissues are highly influenced by the mechanical properties of the extracellular matrix. Stiffness gradient hydrogels are frequently used for exploring these complex relationships in mechanobiology. In this study, the fabrication of a simple, cost‐efficient, and versatile system is reported for creation of stiffness gradients from photoactive hydrogels like gelatin‐methacryloyl (GelMA). The setup includes syringe pumps for gradient generation and a 3D printed microfluidic device for homogenous mixing of GelMA precursors with different crosslinker concentration. The stiffness gradient is investigated by using rheology. A co‐culture consisting of human adipose tissue‐derived mesenchymal stem cells (hAD‐MSCs) and human umbilical cord vein endothelial cells (HUVECs) is encapsulated in the gradient construct. It is possible to locate the stiffness ranges at which the studied cells displayed specific spreading morphology and migration rates. With the help of the described system, variable mechanical gradient constructs can be created and optimal 3D cell culture conditions can be experientially identified.     

Tailoring 3D hydrogel systems for neuronal encapsulation in living electrodes

State‐of‐the‐art neurorprostheses rely on stiff metallic electrodes to communicate with neural tissues. It was envisioned that a soft, organic electrode coating embedded with functional neural cells will enhance electrode‐tissue integration. To enable such a device, it is necessary to produce a cell scaffold with mechanical properties matched to native neural tissue. A degradable poly(vinyl alcohol) (PVA) hydrogel was tailored to have a range of compressive moduli through variation in macromer composition and initiator amount. A regression model was used to predict the amount of initiator required for hydrogel polymerization with nominal macromer content ranging between 5 and 20 wt %. Hydrogels at 5 and 10 wt % were reliably formed but 15 wt % and above were not able to be fabricated due to the light attenuation properties of the initiator ruthenium at increased concentration. Compressive modulus of hydrogels decreased upon incorporation of biomolecules (sericin and gelatin), however, the bulk stiffness spanned the range required to match neural tissue properties (0.04–20kPa). Neuroglia cells, such as Schwann cells survived and grew within the scaffold. The significant finding of this work is that the PVA‐tyramine system can be tuned to provide a soft degradable scaffold for neural tissue regeneration while presenting bioactive molecules for cellular expansion. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 273–287     

Covalent Incorporation of Heparin Improves Chondrogenesis in Photocurable Gelatin‐Methacryloyl Hydrogels

Multicomponent gelatin‐methacryloyl (GelMA) hydrogels are regularly adopted for cartilage tissue engineering (TE) applications, where optimizing chemical modifications for preserving biofunctionality is often overlooked. This study investigates the biological effect of two different modification methods, methacrylation and thiolation, to copolymerize GelMA and heparin. The native bioactivity of methacrylated heparin (HepMA) and thiolated heparin (HepSH) is evaluated via thromboplastin time and heparan sulfate‐deficient myeloid cell‐line proliferation assay, demonstrating that thiolation is superior for preserving anticoagulation and growth factor signaling capacity. Furthermore, incorporating either HepMA or HepSH in chondrocyte‐laden GelMA hydrogels, cultured for 5 weeks under chondrogenic conditions, promotes cell viability and chondrocyte phenotype. However, only GelMA‐HepSH hydrogels yield significantly greater differentiation and matrix deposition in vitro compared to GelMA. This study demonstrates that thiol‐ene chemistry offers a favorable strategy for incorporating bioactives into gelatin hydrogels as compared to methacrylation while furthermore highlighting GelMA‐HepSH hydrogels as candidates for cartilage TE applications.