Shear-thinning Hydrogels Research Articles

Dual physical dynamic bond-based injectable and biodegradable hydrogel for tissue regeneration.

A biodegradable and injectable hydrogel was designed using dual physical dynamic bonds based on host-guest chemistry and electrostatic interactions to build up the network structure. The material was synthesized by simultaneously coupling mono-carboxylic acid terminated poly(ethylene glycol) and arginine to poly(ethylene aspartate diglyceride) to yield a mPEG-grafted poly(ethylene argininylaspartate diglyceride) (mPEG-g-PEAD). When mixing this polymer with α-cyclodextrin and a natural polyanion (heparin), the supramolecular network was formed in a quick gelation with shear thinning properties. The in vitro cytotoxicity was evaluated using primary baboon arterial smooth muscle cells (BaSMCs) and the results showed that cell membrane integrity, viability and metabolism were not compromised by this synthetic polycation at concentrations as high as 10 mg mL-1, a 1000-fold lower toxicity than commercial PEI. The in vitro biocompatibility of the as-made hydrogel was also evaluated using BaSMCs. Neither the hydrogel nor the hydrogel components altered cell behavior in the assays. Fibroblast growth factor 2 was incorporated into the hydrogel and sustainably released at a nearly stable rate up to 16 days without initial burst release, suggesting potential applications in wound healing and ischemic tissue regeneration, among others.

Injectable shear-thinning nanoengineered hydrogels for stem cell delivery.

Injectable hydrogels are investigated for cell encapsulation and delivery as they can shield cells from high shear forces. One of the approaches to obtain injectable hydrogels is to reinforce polymeric networks with high aspect ratio nanoparticles such as two-dimensional (2D) nanomaterials. 2D nanomaterials are an emerging class of ultrathin materials with a high degree of anisotropy and they strongly interact with polymers resulting in the formation of shear-thinning hydrogels. Here, we present 2D nanosilicate reinforced kappa-carrageenan (κCA) hydrogels for cellular delivery. κCA is a natural polysaccharide that resembles native glycosaminoglycans and can form brittle hydrogels via ionic crosslinking. The chemical modification of κCA with photocrosslinkable methacrylate groups renders the formation of a covalently crosslinked network (MκCA). Reinforcing the MκCA with 2D nanosilicates results in shear-thinning characteristics, and enhanced mechanical stiffness, elastomeric properties, and physiological stability. The shear-thinning characteristics of nanocomposite hydrogels are investigated for human mesenchymal stem cell (hMSC) delivery. The hMSCs showed high cell viability after injection and encapsulated cells showed a circular morphology. The proposed shear-thinning nanoengineered hydrogels can be used for cell delivery for cartilage tissue regeneration and 3D bioprinting.

Direct 3D printing of shear-thinning hydrogels at high resolution using open-source technologies

Direct 3D printing of shear-thinning hydrogels at high resolution using open-source technologies

Abstract 17861: Injectable Shear-thinning Hydrogels to Deliver Endothelial Progenitor Cells, Reduce Ventricular Strain, and Dynamic Stiffness to Stabilize Cardiac Function Following Ischemic Injury

The ventricle undergoes adverse remodeling, post-infarction. We developed an injectable shear-thinning hyaluronic acid hydrogel(STG) and endothelial progenitor cell construct(STG-EPC) to enhance vasculogenesis(shown previously) and mechanically stabilize the infarcted myocardium. We hypothesize that this tissue-engineered therapy will minimize adverse remodeling and maintain myocardial tensile properties in a clinically translatable therapy. EPCs(DiLDL+ VEGFR2+ CD34+) were harvested from adult Wistar Rats and suspended in STG. STG-EPC constructs were circumferentially injected at the borderzone of ischemic rat myocardium, following LAD ligation. Engraftment and retention were assessed by near infrared cellular tag. Myocardial remodeling, tensile properties, and hemodynamic function were analyzed in 4 groups: control(PBS), EPC injection(EPC), STG injection(STG), and STG-EPC construct(STG-EPC). High-resolution ultrasound with speckle tracking (VSI Vevo 2100) allowed analysis of regional epicardial and endocardial strain. Uniaxial testing assessed tensile strength. STG-EPC injection significantly increased engraftment, migration, and retention of the EPCs one-week post implant compared to EPC (0.7 vs 0.4 RFU, p=0.02). Using strain echocardiography a significant increase in left ventricular function was noted in the STG-EPC cohort. Strain echocardiography examination revealed a significant retention of myocardial longitudinal displacement with subsequent stabilization of myocardial velocity with STG-EPC therapy. A reduction in dynamic stiffness was noted in the STG-EPC cohort.(Table) This novel injectable shear-thinning hyaluronic acid hydrogel seeded with EPCs demonstrates stabilization of borderzone myocardium with reduction in adverse myocardial remodeling and preservation of myocardial biomechanics. There is a markedly increased retention of delivered stem cells with this tissue engineered therapy.

Abstract 15060: Protein-Engineered Hydrogels for Improved Efficacy of Stem Cell-Based Injection Therapy in a Murine Model of Peripheral Arterial Disease

Introduction: Stem cell injection is a minimally invasive approach for treatment of cardiovascular diseases including peripheral arterial disease (PAD), which affects over 8 million patients in the US. However, poor cell survival and low cell retention at injection site are critical bottlenecks to the efficacy of stem cell therapy. We developed a shear-thinning and self-healing hydrogel system with controllable rigidity for stem cell encapsulation so as to enhance transplant cell viability. Hypothesis: The injectable hydrogel system will prolong cell survival under ischemic conditions and maintain cellular phenotype, in comparison to cell injection in saline. Methods: We developed a hydrogel comprised of two complementary engineered proteins that self-assemble upon simple mixing. The hydrogel network incorporates a polyethylene glycol physical crosslinker that modulates hydrogel stiffness and degradation. Bioluminescently-labeled human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) were encapsulated within the hydrogel with controllable stiffness (G'~10-800 Pa) under conditions of hypoxia (1% O2). The cells within hydrogel were then subjected to an in vitro model of injection and assayed for cell survival, proliferation, and endothelial phenotype for up to 14 days. To verify these results in an experimental model of PAD, 10^6 cells were injected in saline or in 400 Pa hydrogel into the ischemic limb in SCID mice. Results: Acutely after injection, the survival of iPSC-ECs in saline was 65%, whereas survival of iPSC-ECs in hydrogels with stiffnesses of 10, 100, 400, and 800 Pa was 94%. Bioluminescence imaging of cell viability in hypoxia for 14 days demonstrated the highest proliferation in the hydrogel with 400 Pa stiffness. In the 400 Pa hydrogel, iPSC-ECs maintained elongated morphology with robust expression of endothelial phenotypic marker, CD31. In the ischemic hindlimb, iPSC-EC retention was markedly increased with hydrogel encapsulation, compared to saline delivery. Conclusions: These findings demonstrate that stem cell encapsulation within this protein hydrogel improves cell viability, which may have therapeutic benefit for treatment of PAD and broadly to myocardial ischemia.

Injectable shear-thinning hydrogels used to deliver endothelial progenitor cells, enhance cell engraftment, and improve ischemic myocardium

ObjectivesThe clinical translation of cell-based therapies for ischemic heart disease has been limited because of low cell retention (<1%) within, and poor targeting to, ischemic myocardium. To address these issues, we developed an injectable hyaluronic acid (HA) shear-thinning hydrogel (STG) and endothelial progenitor cell (EPC) construct (STG-EPC). The STG assembles as a result of interactions of adamantine- and β-cyclodextrin–modified HA. It is shear-thinning to permit delivery via a syringe, and self-heals upon injection within the ischemic myocardium. This directed therapy to the ischemic myocardial border zone enables direct cell delivery to address adverse remodeling after myocardial infarction. We hypothesize that this system will enhance vasculogenesis to improve myocardial stabilization in the context of a clinically translatable therapy. MethodsEndothelial progenitor cells (DiLDL+ VEGFR2+ CD34+) were harvested from adult male rats, cultured, and suspended in the STG. In vitro viability was quantified using a live-dead stain of EPCs. The STG-EPC constructs were injected at the border zone of ischemic rat myocardium after acute myocardial infarction (left anterior descending coronary artery ligation). The migration of the enhanced green fluorescent proteins from the construct to ischemic myocardium was analyzed using fluorescent microscopy. Vasculogenesis, myocardial remodeling, and hemodynamic function were analyzed in 4 groups: control (phosphate buffered saline injection); intramyocardial injection of EPCs alone; injection of the STG alone; and treatment with the STG-EPC construct. Hemodynamics and ventricular geometry were quantified using echocardiography and Doppler flow analysis. ResultsEndothelial progenitor cells demonstrated viability within the STG. A marked increase in EPC engraftment was observed 1-week postinjection within the treated myocardium with gel delivery, compared with EPC injection alone (17.2 ± 0.8 cells per high power field (HPF) vs 3.5 cells ± 1.3 cells per HPF, P = .0002). A statistically significant increase in vasculogenesis was noted with the STG-EPC construct (15.3 ± 5.8 vessels per HPF), compared with the control (P < .0001), EPC (P < .0001), and STG (P < .0001) groups. Statistically significant improvements in ventricular function, scar fraction, and geometry were noted after STG-EPC treatment compared with the control. ConclusionsA novel injectable shear-thinning HA hydrogel seeded with EPCs enhanced cell retention and vasculogenesis after delivery to ischemic myocardium. This therapy limited adverse myocardial remodeling while preserving contractility.

Selective Proteolytic Degradation of Guest-Host Assembled, Injectable Hyaluronic Acid Hydrogels.

There have been significant advances in the past decades toward the engineering of materials with biomimetic properties. In particular, hydrogels covalently cross-linked with protease degradable peptides have demonstrated the importance of protease mediated degradation for targeted therapeutic cargo delivery and controlling cell-material interactions. However, the incorporation of such degradation mechanisms into synthetic shear-thinning hydrogels has yet to be accomplished. Herein, we utilize supramolecular self-assembly mediated by the guest-host interaction of hyaluronic acid (HA) separately modified by adamantane (Ad) or cyclodextrin (CD) to form shear-thinning and self-healing hydrogels. In this design, Ad is bound to HA via a proteolytically degradable peptide tether (attached via Michael-addition of a cysteine residue in an Ad-terminated peptide with maleimide modified HA), enabling subsequent proteolytic degradation of the assembly. Upon mixing of the Ad-peptide modified HA and the CD modified HA, a supramolecular hydrogel was formed (G' ≈ 300 Pa at 1 Hz), which displayed shear-thinning (>80% viscosity reduction at 0.5 s-1) and near-instantaneous self-healing properties. Rational, selective modification of amino acid residues near the proteolytic site enabled control over peptide cleavage kinetics, specifically with either collagenases or MMP-2. Hydrogel degradation, mediated by a combination of stochastically governed erosion and proteolytic degradation, was influenced by peptide susceptibility to proteolysis both in vitro and in vivo (>2 fold difference at 3 weeks in vivo) when injected subcutaneously. This material system provides unique opportunities for therapeutic delivery (e.g., growth factors, cells) through facile material formation, ease of injection, and bioresponsive material degradation.

Characterization of Carbopol® hydrogel rheology for experimental tectonics and geodynamics

One of the long-standing challenges of modern tectonics and geodynamics is to fully understand the strong strain localization and its effects observed in the lithosphere, which presents viscous, as well as elastic and brittle properties. Recently yield stress–shear thinning hydrogels, such as Carbopol®, have been employed in analog modeling because of its great potential for mimicking the non-Newtonian behavior of rocks. Conversely its use has been limited by the difficulties in assessing its rheology and in preparing uniform samples. Ergo, it is essential to ensure a standard recipe, yielding to a reproducible behavior, no matter which rheometer model is used. We carried out, at four institutions (FAST, GFZ, IPGP and LET), a benchmark for developing a standard preparation and for testing the comparability of results. Then, we conducted a systematical rheological characterization of a wide range of Carbopol® formulas as a function of concentration, composition, pH, temperature and aging. Results show that neutral pH favors higher viscosity. The shear modulus, yield stress, viscosity, and shear thinning behavior increase with concentration. The linear viscoelastic range increases with concentration contrarily to what is observed in gelatins or colloidal suspensions. A weak inverse relationship between temperature and viscosity is found. Similarly, aging reduces both the viscosity and loss modulus, with reduction more evident for low concentration samples. Scaling analysis revealed that low concentration samples, i.e. <0.1wt.%, exhibiting shear thinning behavior and low yield stress, are appropriate to model the rising of thermal instabilities. Those at 0.5–1.0wt.%, showing yield stress in the order of hundreds of Pa and n ranging between 1.6 and 3.4 are good candidates to mimic the non-linear ductile behavior of crustal rocks. We conclude that tuning the visco-elasto-plastic rheology of Carbopol® would make this material a good candidate for modeling of also other geological processes.

Shear-Thinning and Rapid-Recovery Peptide Hydrogel for Biomedical Applications

ABSTRACTPeptides have become attractive molecules for fabricating biomaterials. Studies of peptide structure, assembly properties, and dynamic behavior in response to external parameters have led to rational novel design of peptide biomaterials. One model sequence selected was a β-spiral motif of spider flagelliform silk protein, [GPGGX]n (X = any amino acid). Modifying the X residue can change the quantity of secondary structure and the stability of this spider silk motif. Glycine provides flexible properties, and proline influences the secondary structure and mechanical properties. Another model sequence was GXGXDXUX (U = hydrophobic residue), a Ca2+ binding domain of lipase Lip A from Serratia marcescens, in which aspartate residue is required for ion binding. Combining with [GPGGX]n, we rationally designed peptide as GPGGDGPGGD (eD2). The Ca2+ binding sequence was hidden in the first eight residues of eD2. As expected, this peptide can assemble into nanofibrils triggered by Ca2+ ions. Using the segment FLIVIGSII (h9) from the third trans-membrane segment of subunit IV in the dihydropyridine sensitive human muscle L-type calcium channel as the hydrophobic motif, we obtained FLIVIGSIIGPGGDGPGGD (h9e) peptide. The h9e self-assembled into nanofibrils and further formed shear-thinning and rapid recovery hydrogel in neutral pH range from 6.0 to 8.0 with a large working range of temperature. NMR study showed that amphiphilic structure of h9e peptide tended to adopt a more helical structure during hydrogel formation. The h9e peptide has great potential for biomedical applications. MCF-7 cells were successfully grown as colony-like clusters (reminiscent of real tumors) in h9e hydrogel system. The drug response test of cisplatin further demonstrated the capability of h9e system for drug screen. Moreover, h9e hydrogel showed a promising adjuvanticity by enhancing the vaccine efficacy for killed H1N1 swine influenza virus and PRRS modified live virus.

Rational Design of Network Properties in Guest–Host Assembled and Shear-Thinning Hyaluronic Acid Hydrogels

Shear-thinning hydrogels afford direct injection or catheter delivery to tissues without potential premature gel formation and delivery failure or the use of triggers such as chemical initiators or heat. However, many shear-thinning hydrogels require long reassembly times or exhibit rapid erosion. We developed a shear-thinning hyaluronic acid (HA) hydrogel based on the guest-host interactions of adamantane modified HA (guest macromer, Ad-HA) and β-cyclodextrin modified HA (host macromer, CD-HA). The ability of the guest and host molecules to interact with their counterpart following conjugation to HA was confirmed by (1)H NMR spectroscopy and was similar to that of the native complex. Mixing of Ad-HA and CD-HA resulted in rapid formation of a hydrogel composed of guest-host bonds. The hydrogel physical properties, including mechanics and flow characteristics, were dependent on cross-link density and network structure, which were controlled through macromer concentration, the extent of guest macromer modification, and the molar ratio of guest and host functional groups. The guest-host assembly mechanism permitted both shear-thinning behavior for ease of injection and near-instantaneous reassembly for material retention at the target sight. The hydrogel erosion and release of a model biomolecule were also dependent on design parameters and were sustained for over 60 days. These hydrogels show potential as a minimally invasive injectable hydrogel for biomedical applications.

Self‐Assembly: Reinforcement of Shear Thinning Protein Hydrogels by Responsive Block Copolymer Self‐Assembly (Adv. Funct. Mater. 9/2013)

Triblock copoylmers with associative protein midblocks and thermoresponsive endblocks form shear thinning hydrogels that can be injected at low temperature. On page 1182, Bradley D. Olsen and co-workers report that when warmed to body temperature, the gel is responsively reinforced as the endblocks self-assemble into a secondary block copolymer network, leading to large increases in modulus and yield stress, and decreased erosion and creep compliance.

Secondary Photocrosslinking of Injectable Shear‐Thinning Dock‐and‐Lock Hydrogels

Shear-thinning hydrogels are useful in numerous applications, including as injectable carriers that act as scaffolds to support cell and drug therapies. Here, we describe the engineering of a self-assembling Dock-and-Lock (DnL) system that forms injectable shear-thinning hydrogels using molecular recognition interactions that also possess photo-triggerable secondary crosslinks. These DnL hydrogels are fabricated from peptide-modified hyaluronic acid (HA) and polypeptide precursors, can self-heal immediately after shear induced flow, are cytocompatible, and can be stabilized through light-initiated radical polymerization of methacrylate functional groups to tune gel mechanics and erosion kinetics. Secondary crosslinked hydrogels retain self-adhesive properties and exhibit cooperative physical and chemical crosslinks with moduli as high as ∼10 times larger than moduli of gels based on physical crosslinking alone. The extent of reaction and change in properties are dependent on whether the methacrylate is incorporated either at the terminus of the peptide or directly to the HA backbone. Additionally, the gel erosion can be monitored through an incorporated fluorophore and physical-chemical gels remain intact in solution over months, whereas physical gels that are not covalently crosslinked erode completely within days. Mesenchymal stem cells exhibit increased viability when cultured in physical- chemical gels, compared with those cultured in gels based on physical crosslinks alone. The physical properties of these DnL gels may be additionally tuned by adjusting component compositions, which allows DnL gels with a wide range of physical properties to be constructed for use.

Reinforcement of Shear Thinning Protein Hydrogels by Responsive Block Copolymer Self-Assembly.

Shear thinning hydrogels are promising materials that exhibit rapid self-healing following the cessation of shear, making them attractive for a variety of applications including injectable biomaterials. In this work, self-assembly is demonstrated as a strategy to introduce a reinforcing network within shear thinning artificially engineered protein gels, enabling a responsive transition from an injectable state at low temperatures with a low yield stress to a stiffened state at physiological temperatures with resistance to shear thinning, higher toughness, and reduced erosion rates and creep compliance. Protein-polymer triblock copolymers capable of the responsive self-assembly of two orthogonal networks have been synthesized by conjugating poly(N-isopropylacrylamide) to the N- and C- termini of a protein midblock decorated with coiled-coil self-associating domains. Midblock association forms a shear-thinning network, while endblock aggregation at elevated temperatures introduces a second, independent physical network into the protein hydrogel. These new, reversible crosslinks introduce extremely long relaxation times and lead to a five-fold increase in the elastic modulus, significantly larger than is expected from transient network theory. Thermoresponsive reinforcement reduces the high temperature creep compliance by over four orders of magnitude, decreases the erosion rate by at least a factor of five, and increases the yield stress by up to a factor of seven. The reinforced hydrogels also exhibit enhanced resistance to plastic deformation and failure in uniaxial compression. Combined with the demonstrated potential of shear thinning artificial protein hydrogels for various uses, including the minimally-invasive implantation of bioactive scaffolds, this reinforcement mechanism broadens the range of applications that can be addressed with shear-thinning physical gels.

Shear-thinning hydrogels for biomedical applications

Injectable hydrogels are becoming increasingly important in the fields of tissue engineering and drug delivery due to their tunable properties, controllable degradation, high water content, and the ability to deliver them in a minimally invasive manner. Shear-thinning is one promising technique for the application of injectable hydrogels, where preformed hydrogels can be injected by application of shear stress (during injection) and quickly self-heal after removal of shear. Importantly, these gels can be used to deliver biological molecules and cells during the injection process. This review aims to highlight the range of injectable shear-thinning hydrogel systems being developed, with a focus on the various mechanisms of formation and shear-thinning and their use in biomedical applications.

Injectable shear-thinning hydrogels engineered with a self-assembling Dock-and-Lock mechanism

Injected therapeutics, such as cells or biological molecules, may have enhanced efficiency when delivered within a scaffold carrier. Here, we describe a dual-component Dock-and-Lock (DnL) self-assembly mechanism that can be used to construct shear-thinning, self-healing, and injectable hydrogels. One component is derived from the RIIα subunit of cAMP-dependent kinase A and is engineered as a telechelic protein with end groups that dimerize (docking step). The second component is derived from the anchoring domain of A-kinase anchoring protein (AD) and is attached to multi-arm crosslinker polymers and binds to the docked proteins (locking step). When mixed, these two DnL components form robust physical hydrogels instantaneously and under physiological conditions. Mechanical properties and erosion rates of DnL gels can be tuned through the AD peptide sequence, the concentration and ratio of each component, and the number of peptides on the cross-linking polymer. DnL gels immediately self-recover after deformation, are resistant to yield at strains as high as 400%, and completely self-heal irrespective of prior mechanical disruption. Mesenchymal stem cells mixed in DnL gels and injected through a fine needle remain highly viable (>90%) during the encapsulation and delivery process, and encapsulated large molecules are released with profiles that correspond to gel erosion. Thus, we have used molecular engineering strategies to develop cytocompatible and injectable hydrogels that have the potential to support cell and drug therapies.