Percolation-induced gel-gel phase separation in a dilute polymer network.

Cosmic large-scale structures, animal flocks and living tissues can be considered non-equilibrium organized systems created by dissipative processes. Replicating such properties in artificial systems is still difficult. Herein we report a dissipative network formation process in a dilute polymer-water mixture that leads to percolation-induced gel-gel phase separation. The dilute system, which forms a monophase structure at the percolation threshold, spontaneously separates into two co-continuous gel phases with a submillimetre scale (a dilute-percolated gel) during the deswelling process after the completion of the gelation reaction. The dilute-percolated gel, which contains 99% water, exhibits unexpected hydrophobicity and induces the development of adipose-like tissues in subcutaneous tissues. These findings support the development of dissipative structures with advanced functionalities for distinct applications, ranging from physical chemistry to tissue engineering.

Analysis of the sustained release ability of bevacizumab-loaded tetra-PEG gel.

Multiple intravitreal injections, which are painful and costly, are often required in the treatment of retinal disorders. Therefore, a novel drug delivery system using hydrogels is currently being evaluated as an alternative. This study aimed to evaluate the ability of tetra-armed polyethylene glycol (tetra-PEG) gel for sustained release in vitro. Bevacizumab-loaded tetra-PEG gel and 5-Carboxyfluorescein N-succinimidyl ester (FAM-NHS)-labeled IgG-loaded tetra-PEG gel were prepared by mixing tetra-PEG with thiol termini (tetra-PEG-SH) solution, maleimide termini (tetra-PEG-MA) solution, and bevacizumab or FAM-NHS labeled IgG. The gels were prepared with three different polymer concentrations of 1.5%, 5%, and 10%, then an in vitro release study performed to assess the sustained release ability of the drug-loaded tetra-PEG gels. High performance liquid chromatography (HPLC) was used to test the structural stability of the bevacizumab released from the tetra-PEG gel. The binding of bevacizumab to tetra-PEG-SH or MA was assessed using SDS-polyacrylamide gel electrophoresis (PAGE). The bioactivity of released bevacizumab was tested using KDR/NFAT-RE HEK293 cells. In addition, in vitro degradation and swelling studies were also performed. The in vitro release analysis showed that the release of bevacizumab was slower in the 5% and 10% tetra-PEG gels than that of 1.5% tetra-PEG gels. Similarly, the release of FAM-NHS-labeled IgG was slowest in the 1.5%, 5%, and 10% tetra-PEG gels, in that order. The 5% and 10% tetra-PEG gels released bevacizumab and FAM-NHS-labeled IgG over a period of 1-2 weeks. Both bevacizumab and FAM-NHS-labeled IgG were not fully released in 2 weeks. HPLC analysis showed that the retention time of the samples released from the bevacizumab-loaded tetra-PEG gel was similar to that of the bevacizumab standard. The SDS-PAGE analysis showed that bevacizumab binds to tetra-PEG-MA. The bioactivity assay test revealed no decrease in the bioactivity of the released bevacizumab. In vitro degradation and swelling studies revealed that 1.5%, 5%, and 10% tetra-PEG gels expanded by approximately 1.4-, 2-, and 3-fold, respectively. Based on the results of the release and swelling tests, 5% tetra-PEG gels are considered good candidates for controlled release systems for therapeutic antibodies such as bevacizumab. The binding of PEG to the therapeutic antibodies may reduce the availability of therapeutic antibodies that can be released.

Hemostatic Capability of a Novel Tetra-Polyethylene Glycol Hydrogel.

BACKGROUND: TetraStat is a tetra-armed polyethylene glycol (PEG) hydrogel. It is a synthetic sealant that solidifies instantly in response to pH changes. This study aimed to evaluate the hemostatic effect of TetraStat through experiments evaluating future clinical applications. METHODS: We used TetraStat, oxidized regenerated cellulose (SURGICEL®), and fibrinogen and thrombin sealant patch (TachoSil®) using in vitro and in vivo experiments. For the in vitro experiment, a closed circulatory system filled with phosphate-buffered saline under high pressure was used. Needle punctures were created and closed using the various sealants. For the in vivo experiment, rat venae cavae were punctured with 18- and 20-gauge (G) needles, and hemorrhage was allowed to occur for several seconds. A porous PEG sponge soaked with TetraStat was applied as a hemostatic system. Hemostasis outcomes were compared among the various concentrations (40-100 g/L) of TetraStat, SURGICEL, and TachoSil. RESULTS: The punctured holes in the prosthetic graft were successfully sealed with TetraStat in 1 min. The success rate of hemostasis with TetraStat for the punctured holes in the rat vena cava was dose-dependent. TetraStat was effective in sealing the holes created with a 20 G needle at all concentrations; however, the holes created with an 18 G needle could be sealed only when the concentration ≥60 g/L. Hemostasis using SURGICEL or TachoSil was less successful and sometimes required up to 5 min. CONCLUSIONS: TetraStat has a high hemostatic ability. A porous PEG sponge soaked with TetraStat is a useful choice for effective hemostasis during massive hemorrhage.

Enzyme-Catalyzed Bottom-Up Synthesis of Mechanically and Physicochemically Stable Cellulose Hydrogels for Spatial Immobilization of Functional Colloidal Particles.

The dispersion stabilization of colloidal particles and subsequent construction of functional materials are of great interest in areas ranging from colloid chemistry to materials science. A promising strategy is the spatial immobilization of colloidal particles within gel scaffolds. However, conventional gels readily deform and even collapse when changes in environmental conditions occur. Herein, we describe the enzyme-catalyzed bottom-up synthesis of mechanically and physicochemically stable nanoribbon network hydrogels composed of crystalline cellulose oligomers in which cellulose nanocrystals (CNCs) as model colloidal particles are immobilized spatially. The stiffness of the hydrogels increased with the amount of CNCs incorporated. Filling the void space of the hydrogels with hydrophobic polymers resulted in polymer nanocomposites with excellent mechanical properties. The nanoribbon networks will be useful for demonstrating the potential functions of colloidal particles.

Slope-Dependent Cell Motility Enhancements at the Walls of PEG-Hydrogel Microgroove Structures.

In recent years, research utilizing micro- and nanoscale geometries and structures on biomaterials to manipulate cellular behaviors, such as differentiation, proliferation, survival, and motility, have gained much popularity; however, how the surface microtopography of 3D objects, such as implantable devices, can affect these various cell behaviors still remains largely unknown. In this study, we discuss how the walls of microgroove topography can influence the morphology and the motility of unrestrained cells, in a different fashion from 2D line micropatterns. Here adhesive substrates made of tetra(polyethylene glycol) (tetra-PEG) hydrogels with microgroove structures or 2D line micropatterns were fabricated, and cell motility on these substrates was evaluated. Interestingly, despite being unconstrained, the cells exhibited drastically different migration behaviors at the edges of the 2D micropatterns and the walls of microgroove structures. In addition to acquiring a unilamellar morphology, the cells increased their motility by roughly 3-fold on the microgroove structures, compared with the 2D counterpart or the nonpatterned surface. Immunostaining revealed that this behavior was dependent on the alignment and the aggregation of the actin filaments, and by varying the slope of the microgroove walls, it was found that relatively upright walls are necessary for this cell morphology alterations. Further progress in this research will not only deepen our understanding of topography-assisted biological phenomena like cancer metastasis but also enable precise, topography-guided manipulation of cell motility for applications such as cancer diagnosis and cell sorting.

Probing the cross-effect of strains in non-linear elasticity of nearly regular polymer networks by pure shear deformation.

The pure shear deformation of the Tetra-polyethylene glycol gels reveals the presence of an explicit cross-effect of strains in the strain energy density function even for the polymer networks with nearly regular structure including no appreciable amount of structural defect such as trapped entanglement. This result is in contrast to the expectation of the classical Gaussian network model (Neo Hookean model), i.e., the vanishing of the cross effect in regular networks with no trapped entanglement. The results show that (1) the cross effect of strains is not dependent on the network-strand length; (2) the cross effect is not affected by the presence of non-network strands; (3) the cross effect is proportional to the network polymer concentration including both elastically effective and ineffective strands; (4) no cross effect is expected exclusively in zero limit of network concentration in real polymer networks. These features indicate that the real polymer networks with regular network structures have an explicit cross-effect of strains, which originates from some interaction between network strands (other than entanglement effect) such as nematic interaction, topological interaction, and excluded volume interaction.

Electrophoretic mobility of semi-flexible double-stranded DNA in defect-controlled polymer networks: Mechanism investigation and role of structural parameters.

Our previous studies have reported an empirical model, which explains the electrophoretic mobility (µ) of double-stranded DNA (dsDNA) as a combination of a basic migration term (Rouse-like or reptation) and entropy loss term in polymer gels with ideal network structure. However, this case is of exception, considering a large amount of heterogeneity in the conventional polymer gels. In this study, we systematically tune the heterogeneity in the polymer gels and study the migration of dsDNA in these gels. Our experimental data well agree with the model found for ideal networks. The basic migration mechanism (Rouse-like or reptation) persists perfectly in the conventional heterogeneous polymer gel system, while the entropy loss term continuously changes with increase in the heterogeneity. Furthermore, we found that in the limit where dsDNA is shorter than dsDNA persistence length, the entropy loss term may be related to the collisional motions between DNA fragments and the cross-links.

Experimental verification of fracture mechanism for polymer gels with controlled network structure.

Recently, polymer gels have drawn much attention as scaffolds for regenerative medicines, soft actuators, and functional membranes. These applications need tough and robust polymer gels as represented by the double network gels. To fully understand this mechanism and develop further advanced polymer gels, we need to fully understand the molecular origin of fracture energy for conventional polymer gels, which is inhibited by the inherent heterogeneity. In this paper, we show the experimental results on the fracture of model polymer gels with controlled network structure, and discuss the mechanism of the fracture of polymer gels.

A computer simulation of the networked structure of a hydrogel prepared from a tetra-armed star pre-polymer.

We used a coarse-grained (CG) molecular dynamics model with potentials convertible to actual units to simulate the polymerization of a gel of a tetra-armed poly(ethylene glycol) derivative (MW ≈ 6000) under aqueous conditions and analysed its three-dimensional network structure. The radius of gyration of individual pre-polymers after gelation was slightly increased compared with that of the single pre-polymer before gelation, and its distribution was broad, attributable to inter- and intra-molecular bonds. The largest pores in the unit cell were about 3.5-3.9 nm. The existence of large pores seems to explain the protein encapsulation capability of and protein leakage from the gel indicating that the CG simulation, which maintains information about potentials in actual units, is an effective tool for investigating gel properties that are difficult to measure in real experiments.

Mechanical properties of tetra-PEG gels with supercoiled network structure.

We investigate the effects of swelling and deswelling on the mechanical properties of tetra-polyethylene glycol gels with the precisely tuned polymerization degree of network strand (Nc) and polymer volume fraction at preparation (ϕ0) by varying the fraction of interest (ϕm). The ϕm-dependence of the elastic modulus exhibits a crossover at ϕc due to large contraction of the network strands (supercoiling) accompanying deswelling. The Obukhov model successfully describes the ϕm-dependence of the elastic modulus. We estimate the fractal dimension of network strands (Df) by analyzing the stress-elongation relationships at high stretching using Pincus blob concept. In the supercoiling region, Df increases with an increase in ϕm, which suggests that the gyration radius of network strands decreases with deswelling in affine manner. The extensibility increases with an increase in ϕm because the deswelling reduces the distance between the neighboring junctions. These findings will help to understand the structure and formation mechanism of supercoiling.

Comparison of the long-term effects on rabbit bone defects between Tetrabone and ß-tricalcium phosphate granules implantation.

Tetrabone is a newly developed granular artificial bone. The 1-mm Tetrabone has a four-legged structure. In this study, the long-term effect of implanting Tetrabone or ß-TCP granules in rabbit femoral cylindrical defects was evaluated. The rabbits were euthanized at 4, 13, and 26 weeks after implantation. Micro-CT was conducted to evaluate the residual material volume and the non-osseous tissue volume. New bone tissue areas were measured by histological analysis. Micro-CT imaging showed that the residual material volume in the ß-TCP group had decreased significantly at 4 weeks after implantation (P < 0.05) and that the ß-TCP granules had nearly disappeared at 26 weeks after implantation. In the Tetrabone group, it did not significantly change until 13 weeks after implantation; it then continued to decrease slightly until 26 weeks after implantation. The non-osseous volume increased in the ß-TCP group, whereas that of the Tetrabone group decreased (P < 0.05). Histological examination showed that the new bone areas were significantly greater in the Tetrabone group than in the ß-TCP group at 13 and 26 weeks. In conclusion, resorption of ß-TCP granules occurs before sufficient bone formation, thereby allowing non-osseous tissue invasion. Tetrabone resorption progressed slowly while the new bone tissues were formed, thus allowing better healing. Tetrabone showed better osteoconductivity, whereas the ß-TCP granules lost their function over a long duration. These results may be caused by the differences in the absorption rate of the granules, intergranular pore structure, and crystallinity of each granule.

Enhancing cell adhesion in synthetic hydrogels via physical confinement of peptide-functionalized polymer clusters.

Artificially synthesized poly(ethylene glycol) (PEG)-based hydrogels are extensively utilized as biomaterials for tissue scaffolds and cell culture matrices due to their non-protein adsorbing properties. Although these hydrogels are inherently non-cell-adhesive, advancements in modifying polymer networks with functional peptides have led to PEG hydrogels with diverse functionalities, such as cell adhesion and angiogenesis. However, traditional methods of incorporating additives into hydrogel networks often result in the capping of crosslinking points with heterogeneous substances, potentially impairing mechanical properties and obscuring the causal relationships of biological functions. This study introduces polymer additives designed to resist prolonged elution from hydrogels, providing a novel approach to facilitate cell culture on non-adhesive surfaces. By clustering tetra-branched PEG to form ultra-high molecular weight hyper-branched structures and functionalizing their termini with cell-adhesive peptides, we successfully entrapped these clusters within the hydrogel matrix without compromising mechanical strength. This method has enabled successful cell culture on inherently non-adhesive PEG hydrogel surfaces at high peptide densities, a feat challenging to achieve with conventional means. The approach proposed in this study not only paves the way for new possibilities with polymer additives but also serves as a new design paradigm for cell culturing on non-cell-adhesive hydrogels.

Preclustering Gelatin for Faster-Forming Injectable Hydrogels: A Strategy for Fabricating 3D Hydrogel Scaffolds with Improved Cell Dispersion.

Gelatin-based injectable hydrogels capable of encapsulating cells are pivotal in tissue engineering because they can conform to any geometry and exhibit physical properties similar to those of living tissues. However, the slow gelation process observed in these cell-encapsulating hydrogels often causes an uneven dispersion of cells. This study proposes an approach for achieving fast gelation of unmodified gelatin under physiological conditions through gelatin preclustering. By using tetrafunctional succinimidyl-terminated poly(ethylene glycol) as a clustering agent, the gelation process is successfully expedited fivefold without requiring chemical modifications, effectively addressing the associated challenges of uneven cell distribution.

Gel-Gel Phase Separation in Clustered Poly(ethylene glycol) Hydrogel with Enhanced Hydrophobicity.

The development of hydrophobic poly(ethylene glycol) (PEG) hydrogels, which are typically hydrophilic, could significantly enhance their application as artificial extracellular matrices. In this study, we designed PEG hydrogels with enhanced hydrophobicity through gel-gel phase separation (GGPS), a phenomenon that uniquely enhances hydrophobicity under ambient conditions, and we elucidated the pivotal role of elasticity in this process. We hypothesized that increased elasticity would amplify GGPS, thereby improving the hydrophobicity and cell adhesion on PEG hydrogel surfaces, despite their inherent hydrophilicity. To test this hypothesis, we engineered dilute oligo-PEG gels via a two-step process, creating polymer networks from tetra-PEG clusters with multiple reaction points. These oligo-PEG gels exhibited significantly higher elasticity, turbidity, and shrinkage upon water immersion compared to dilute PEG gels. Detailed characterization through confocal laser scanning microscopy, rheological measurements, and cell adhesion assays revealed distinct biphasic structures, increased hydrophobicity, and enhanced cell attachability in the dilute oligo-PEG gels. Our findings confirm that elasticity is crucial for effective GGPS, providing a novel method for tailoring hydrogel properties without chemical modification. This research introduces a new paradigm for designing biomaterials with improved cell-scaffolding capabilities, offering significant potential for tissue engineering and regenerative medicine.

Thermoresponsive synthetic polymer-microtubule hybrids.

Thermoresponsive hybrids consisting of synthetic polymers and microtubules (MTs), i.e., assemblies of tubulins, were prepared by bonding MTs covalently to a few reactive units in a macromolecular strand. The hybrids exhibited the gel/sol transition because of the "assembling of tubulins to MTs/disintegrating of MTs to tubulins" by the temperature change between 37 and 4 °C, respectively. The viscoelastic behaviors of the hybrid gels depended upon the quantity of polymer feed and the amount of resulting covalent bonds between the polymers and tubulin units. Furthermore, in a confined space of a thin and long rectangular cell with the temperature gradient from 4 °C (cold terminal) to 37 °C (warm terminal), the sol state hybrid turned to the gel state that propagated from the warm terminal toward the cold terminal to form uniaxially oriented MT arrays. Upon changing the temperature of the whole system between 37 and 4 °C, the uniaxial arrays appeared/disappeared reversibly.

Fracture energy of polymer gels with controlled network structures.

We have investigated the fracture behaviors of tetra-arm polyethylene glycol (Tetra-PEG) gels with controlled network structures. Tetra-PEG gels were prepared by AB-type crosslink-coupling of mutually reactive tetra-arm prepolymers with different concentrations and molecular weights. This series of controlled network structures, for the first time, enabled us to quantitatively examine the Lake-Thomas model, which is the most popular model predicting fracture energies of elastomers. The experimental data showed good agreement with the Lake-Thomas model, and indicated a new molecular interpretation for the displacement length (L), the area around a crack tip within which the network strands are fully stretched. L corresponded to the three times of end-to-end distance of network strands, regardless of all parameters examined. We conclude that the Lake-Thomas model can quantitatively predict the fracture energy of polymer network without trapped entanglements, with the enhancement factor being near 3.

One-Pot Approach to Synthesize Tough and Cell Adhesive Double-Network Hydrogels Consisting of Fully Synthetic Materials of Self-Assembling Peptide and Poly(ethylene glycol).

Hydrogels with a double network (DN) structure are compelling biomaterials, holding potential for use as artificial extracellular matrices. Generally, the DN approach imparts hydrogels with high mechanical strength and cell-adhesive properties. However, achieving this often demands a complex multistep process involving potentially hazardous free-radical polymerization, which can result in toxicity. This limits their broad biological applications. In this work, we introduce a straightforward yet biocompatible method to fabricate tough and cell-adhesive DN hydrogels using entirely synthetic materials: the self-assembling peptide (RADA16) and poly(ethylene glycol) (PEG). An in situ mixing of these components leads to the sequential formation of DN hydrogels─first through the self-assembly of the RADA16 peptide and then via chemical cross-linking between PEG molecules. Hydrogels produced this way exhibited up to a 10-fold increase in fracture energy, and cells seeded on their surfaces showcased good attachment. Our design underscores the efficacy of the DN approach and the promising applications of peptides in tissue engineering.

Tissue-Adhesive Hydrogel Spray System for Live Cell Immobilization on Biological Surfaces.

Gelatin hydrogels are used as three-dimensional cell scaffolds and can be prepared using various methods. One widely accepted approach involves crosslinking gelatin amino groups with poly(ethylene glycol) (PEG) modified with N-hydroxysuccinimide ester (PEG-NHS). This method enables the encapsulation of live cells within the hydrogels and also facilitates the adhesion of the hydrogel to biological tissues by crosslinking their surface amino groups. Consequently, these hydrogels are valuable tools for immobilizing cells that secrete beneficial substances in vivo. However, the application of gelatin hydrogels is limited due to the requirement for several minutes to solidify under conditions of neutral pH and polymer concentrations suitable for live cells. This limitation makes it impractical for use with biological tissues, which have complex shapes or inclined surfaces, restricting its application to semi-closed spaces. In this study, we propose a tissue-adhesive hydrogel that can be sprayed and immobilized with live cells on biological tissue surfaces. This hydrogel system combines two components: (1) gelatin/PEG-NHS hydrogels and (2) instantaneously solidifying PEG hydrogels. The sprayed hydrogel solidified within 5 s after dispensing while maintaining the adhesive properties of the PEG-NHS component. The resulting hydrogels exhibited protein permeability, and the viability of encapsulated human mesenchymal stem/stromal cells (hMSCs) remained above 90% for at least 7 days. This developed hydrogel system represents a promising approach for immobilizing live cells on tissue surfaces with complex shapes.

Molecular Weight-Dependent Diffusion, Biodistribution, and Clearance Behavior of Tetra-Armed Poly(ethylene glycol) Subcutaneously Injected into the Back of Mice.

Four-armed poly(ethylene glycol) (PEG)s are essential hydrophilic polymers extensively utilized to prepare PEG hydrogels, which are valuable tissue scaffolds. When hydrogels are used in vivo, they eventually dissociate due to cleavage of the backbone structure. When the cleavage occurs at the cross-linking point, the hydrogel elutes as an original polymer unit, i.e., four-armed PEG. Although four-armed PEGs have been utilized as subcutaneously implanted biomaterials, the diffusion, biodistribution, and clearance behavior of four-armed PEG from the skin are not fully understood. This paper investigates time-wise diffusion from the skin, biodistribution to distant organs, and clearance of fluorescence-labeled four-armed PEGs with molecular weight (Mw) ranging from 5-40 kg/mol subcutaneously injected into the back of mice. Changes over time indicated that the fate of subcutaneously injected PEGs is Mw-dependent. Four-armed PEGs with Mw ≤ 10 kg/mol gradually diffused to deep adipose tissue beneath the injection site and distributed dominantly to distant organs, such as the kidney. PEGs with Mw ≥ 20 kg/mol stagnated in the skin and deep adipose tissue and were mainly delivered to the heart, lung, and liver. The fundamental understanding of the Mw-dependent behavior of four-armed PEGs is beneficial for preparing biomaterials using PEGs, providing a reference in the field of tissue engineering.

In situ-formable, dynamic crosslinked poly(ethylene glycol) carrier for localized adeno-associated virus infection and reduced off-target effects.

The adeno-associated virus (AAV) is a potent vector for in vivo gene transduction and local therapeutic applications of AAVs, such as for skin ulcers, are expected. Localization of gene expression is important for the safety and efficiency of genetic therapies. We hypothesized that gene expression could be localized by designing biomaterials using poly(ethylene glycol) (PEG) as a carrier. Here we show one of the designed PEG carriers effectively localized gene expression on the ulcer surface and reduced off-target effects in the deep skin layer and the liver, as a representative organ to assess distant off-target effects, using a mouse skin ulcer model. The dissolution dynamics resulted in localization of the AAV gene transduction. The designed PEG carrier may be useful for in vivo gene therapies using AAVs, especially for localized expression.