Gluing blood into gel by electrostatic interaction using a water-soluble polymer as an embolic agent.

Liquid embolic agents are widely used for the endovascular embolization of vascular conditions. However, embolization based on phase transition is limited by the adhesion of the microcatheter to the embolic agent, use of an organic solvent, unintentional catheter retention, and other complications. By mimicking thrombus formation, a water-soluble polymer that rapidly glues blood into a gel without triggering coagulation was developed. The polymer, which consists of cationic and aromatic residues with adjacent sequences, shows electrostatic adhesion with negatively charged blood substances in a physiological environment, while common polycations cannot. Aqueous polymer solutions are injectable through clinical microcatheters and needles. The formed blood gel neither adhered to the catheter nor blocked the port. Postoperative computed tomography imaging showed that the polymer can block the rat femoral artery in vivo and remain at the injection site without nontarget embolization. This study provides an alternative for the development of waterborne embolic agents.

Fractographic mirror law for brittle fracture of nonlinear elastic soft materials.

Mirror radius analysis of fractured surfaces is a powerful fractographic method for determining the cause of failure in linear elastic hard materials because it does not require prior loading information. However, mirror analysis for soft materials is lacking. In this study, we established a general mirror radius law for nonlinear elastic soft materials using highly deformable brittle hydrogels. The fracture stress and mirror radius follow a -1 power law, which differs from the -0.5 power law for linear elastic hard materials. The constant in the power law is related to the fracture energy of the material. This discovery provides insights into fracture mechanisms and leads the way for applying fractography to soft materials.

Mechanical Properties of a 3 Dimensional-Printed Transparent Flexible Resin Used for Vascular Model Simulation Compared with Those of Porcine Arteries.

PURPOSE: To develop a vascular intervention simulation model that replicates the characteristics of a human patient and to compare the mechanical properties of a 3-dimensional (3D)-printed transparent flexible resin with those of porcine arteries using the elastic modulus (E) and kinetic friction coefficient (µk). MATERIALS AND METHODS: Resin plates were created from a transparent flexible resin using a 3D printer. Porcine artery plates were prepared by excising the aorta. E values and the adhesive strengths of the resin and arterial surfaces toward a polyethylene plate, were measured with a tensile-compressive mechanical tester. Resin transparency was measured using an ultraviolet-visible light spectrometer. The µk value of the resin plate surface after applying silicone spray for 1-5 seconds and that of the artery were measured using a translational friction tester. RESULTS: E values differed significantly between the arteries and resin plates at each curing time (0.20 MPa ± 0.04 vs 8.53 MPa ± 2.37 for a curing time of 1 minute; P < .05). The resin was stiffer than the arteries, regardless of the curing times. The visible light transmittance and adhesive strength of the resin decreased as the curing time increased. The adhesive strength of the artery was the lowest. The µk value of the silicone-coated resin surface created by applying silicone for 2-3 seconds (thickness of the silicone layer, 1.6-2.0 µm) was comparable with that of the artery, indicating that the coating imparted a similar slippage to the resin as to the living artery. CONCLUSIONS: A transparent flexible resin is useful for creating a transparent and slippery vascular model for vascular intervention simulation.

Chitin-Based Double-Network Hydrogel as Potential Superficial Soft-Tissue-Repairing Materials.

Chitin is a biopolymer, which has been proven to be a biomedical material candidate, yet the weak mechanical properties seriously limit their potentials. In this work, a chitin-based double-network (DN) hydrogel has been designed as a potential superficial repairing material. The hydrogel was synthesized through a double-network (DN) strategy composing hybrid regenerated chitin nanofiber (RCN)-poly (ethylene glycol diglycidyl ether) (PEGDE) as the first network and polyacrylamide (PAAm) as the second network. The hybrid RCN-PEGDE/PAAm DN hydrogel was strong and tough, possessing Young's modulus (elasticity) E 0.097 ± 0.020 MPa, fracture stress σf 0.449 ± 0.025 MPa, and work of fracture Wf 5.75 ± 0.35 MJ·m-3. The obtained DN hydrogel was strong enough for surgical requirements in the usage of soft tissue scaffolds. In addition, chitin endowed the DN hydrogel with good bacterial resistance and accelerated fibroblast proliferation, which increased the NIH3T3 cell number by nearly five times within 3 days. Subcutaneous implantation studies showed that the DN hydrogel did not induce inflammation after 4 weeks, suggesting a good biosafety in vivo. These results indicated that the hybrid RCN-PEGDE/PAAm DN hydrogel had great prospect as a rapid soft-tissue-repairing material.

Surface charge dominated protein absorption on hydrogels.

Soft tissue engineering requires antifouling materials that are biocompatible and mechanically flexible. Conventional hydrogels containing more than 70 wt% water are thus promising antifouling material candidates. However, some hydrogels are difficult to apply in internal body organs because of undesirable protein absorption on their surfaces. Due to the lack of an effective method for observing the true charge densities of hydrogels, the reason why electrostatic interactions dominate protein absorption behavior remains unclear. In this work, we adopt the microelectrode technique (MET) to study the electrical potentials of hydrogels with negative, positive, and neutral potentials and demonstrate the protein absorption behaviors on those hydrogels. The results show that MET is an effective method to obtain the surface charge densities of various hydrogels. Furthermore, the amounts of absorbed proteins on the gels were quantified with respect to the charge densities of the hydrogels. The results indicate that electrostatic absorption is quantitatively dominated by a combination of hydrogel charge density and overall protein charge. Based on the knowledge obtained in this work, the effects of hydrogel surface charges on protein absorption can be better understood. Thus, the results are expected to promote the application of hydrogels in tissue engineering.

Stretching-induced ion complexation in physical polyampholyte hydrogels.

Recently, we have developed a series of charge balanced polyampholyte (PA) physical hydrogels by random copolymerization in water, which show extraordinarily high toughness, self-healing ability and viscoelasticity. The excellent performance of PA hydrogels is ascribed to dynamic ionic bond formation through inter- and intra-chain interactions. The randomness results in ionic bonds of wide strength distribution, the strong bonds, which serve as permanent crosslinking, imparting the elasticity, while the weak bonds reversibly break and re-form, dissipating energy. In this work, we developed a simple physical method, called a pre-stretching method, to promote the performance of PA hydrogels. By imposing a pre-stretching on the sample in the as-prepared state, ion complexation during dialysis is prominently accelerated and the final performance is largely promoted. Further analysis suggests that the strong bond formation induced by pre-stretching is responsible for the change in final performance. Pre-stretching decreases the entropy of the system and increases the chain alignment, resulting in an increased possibility for strong bond formation.

Coupled instabilities of surface crease and bulk bending during fast free swelling of hydrogels.

Most studies on hydrogel swelling instability have been focused on a constrained boundary condition. In this paper, we studied the mechanical instability of a piece of disc-shaped hydrogel during free swelling. The fast swelling of the gel induces two swelling mismatches; a surface-inner layer mismatch and an annulus-disc mismatch, which lead to the formation of a surface crease pattern and a saddle-like bulk bending, respectively. For the first time, a stripe-like surface crease that is at a right angle on the two surfaces of the gel was observed. This stripe pattern is related to the mechanical coupling of surface instability and bulk bending, which is justified by investigating the swelling-induced surface pattern on thin hydrogel sheets fixed onto a saddle-shaped substrate prior to swelling. A theoretical mechanism based on an energy model was developed to show an anisotropic stripe-like surface crease pattern on a saddle-shaped surface. These results might be helpful to develop novel strategies for controlling crease patterns on soft and wet materials by changing their three-dimensional shape.

Nanoscale TEM Imaging of Hydrogel Network Architecture.

In this work, the authors succeed in direct visualization of the network structure of synthetic hydrogels with transmission electron microscopy (TEM) by developing a novel staining and network fixation method. Such a direct visualization is not carried out because sample preparation and obtaining sufficient contrast are challenging for these soft materials. TEM images reveal robust heterogeneous network architectures at mesh size scale and defects at micro-scale. TEM images also reveal the presence of abundant dangling chains on the surface of the hydrogel network. The real space structural information provides a comprehensive perspective that links bulk properties with a nanoscale network structure, including fracture, adhesion, sliding friction, and lubrication. The presented method has the potential to advance the field.

Engineering of an electrically charged hydrogel implanted into a traumatic brain injury model for stepwise neuronal tissue reconstruction.

Neural regeneration is extremely difficult to achieve. In traumatic brain injuries, the loss of brain parenchyma volume hinders neural regeneration. In this study, neuronal tissue engineering was performed by using electrically charged hydrogels composed of cationic and anionic monomers in a 1:1 ratio (C1A1 hydrogel), which served as an effective scaffold for the attachment of neural stem cells (NSCs). In the 3D environment of porous C1A1 hydrogels engineered by the cryogelation technique, NSCs differentiated into neuroglial cells. The C1A1 porous hydrogel was implanted into brain defects in a mouse traumatic damage model. The VEGF-immersed C1A1 porous hydrogel promoted host-derived vascular network formation together with the infiltration of macrophages/microglia and astrocytes into the gel. Furthermore, the stepwise transplantation of GFP-labeled NSCs supported differentiation towards glial and neuronal cells. Therefore, this two-step method for neural regeneration may become a new approach for therapeutic brain tissue reconstruction after brain damage in the future.

TiO2 synthesis inspired by biomineralization: control of morphology, crystal phase, and light-use efficiency in a single process.

Hydroxyapatite is mineralized along the long axis of collagen fiber during osteogenesis. Mimicking such biomineralization has great potential to control inorganic structures and is fast becoming an important next-generation inorganic synthesis method. Inorganic matter synthesized by biomineralization can have beautiful and functional structures that cannot be created artificially. In this study, we applied biomineralization to the synthesis of the only photocatalyst in practical use today, titanium dioxide (TiO(2)). The photocatalytic activity of TiO(2) mainly relates to three properties: morphology, crystal phase, and light-use efficiency. To optimize TiO(2) morphology, we used a simple sequential peptide as an organic template. TiO(2) mineralized by a ß-sheet peptide nanofiber template forms fiber-like shapes that are not observed for mineralization by peptides in the shape of random coils. To optimize TiO(2) crystal phase, we mineralized TiO(2) with the template at 400 °C to transform it into the rutile phase and at 700 °C to transform it into a mixed phase of anatase and rutile. To optimize light-use efficiency, we introduced nitrogen atoms of the peptide into the TiO(2) structure as doped elemental material during sintering. Thus, this biomineralization method enables control of inorganic morphology, crystal phase, and light-use efficiency in a single process.

Evaluation of biological responses to micro-particles derived from a double network hydrogel.

Double network hydrogels (DN gels) composed of poly (2-acrylamido-2-methyl propanesulfonic acid) (PAMPS) as the brittle first network and poly (N,N-dimethylacrylamide) (PDMA) as the ductile second network have been proven to be a substitute biomaterial for cartilage, with promising biocompatibility and low toxicity, when they are used as bulk materials. For their further applications as articular cartilages, it is essential to understand the biological reactions and adverse events that might be initiated by wear particles derived from these materials. In this study, we used DN gel micro-particles of sizes 4 µm and 10 µm generated by the grinding method to mimic wearing debris of DN gels. The biological responses to particles were then evaluated in a macrophage-cultured system and an inflammatory osteolysis murine model. Our results demonstrated that DN gel particles have the ability to activate macrophages and promote the expression of Tnf-α, both in vitro and in vivo. Furthermore, the implantation of these particles onto calvarial bone triggered local inflammation and bone loss in a mouse model. Our data reveal that the potential foreign body responses to the generated particles from artificial cartilage should receive more attention in artificial cartilage engineering with the goal of developing a safer biocompatible substitute.

ERK MAP Kinase Signaling Regulates RAR Signaling to Confer Retinoid Resistance on Breast Cancer Cells.

Retinoic acid (RA) and its synthetic derivatives, retinoids, have been established as promising anticancer agents based on their ability to regulate cell proliferation and survival. Clinical trials, however, have revealed that cancer cells often acquire resistance to retinoid therapy. Therefore, elucidation of underlying mechanisms of retinoid resistance has been considered key to developing more effective use of retinoids in cancer treatment. In this study, we show that constitutive activation of ERK MAP kinase signaling, which is often caused by oncogenic mutations in RAS or RAF genes, suppresses RA receptor (RAR) signaling in breast cancer cells. We show that activation of the ERK pathway suppresses, whereas its inhibition promotes, RA-induced transcriptional activation of RAR and the resultant upregulation of RAR-target genes in breast cancer cells. Importantly, ERK inhibition potentiates the tumor-suppressive activity of RA in breast cancer cells. Moreover, we also reveal that suppression of RAR signaling and activation of ERK signaling are associated with poor prognoses in breast cancer patients and represent hallmarks of specific subtypes of breast cancers, such as basal-like, HER2-enriched and luminal B. These results indicate that ERK-dependent suppression of RAR activity underlies retinoid resistance and is associated with cancer subtypes and patient prognosis in breast cancers.

Hydroxyapatite-hybridized double-network hydrogel surface enhances differentiation of bone marrow-derived mesenchymal stem cells to osteogenic cells.

Recently, we have developed a hydroxyapatite (HAp)-hybridized double-network (DN) hydrogel (HAp/DN gel), which can robustly bond to the bone tissue in the living body. The purpose of this study is to clarify whether the HAp/DN gel surface can differentiate the bone marrow-derived mesenchymal stem cells (MSCs) to osteogenic cells. We used the MSCs which were harvested from the rabbit bone marrow and cultured on the polystyrene (PS) dish using the autogenous serum-supplemented medium. First, we confirmed the properties of MSCs by evaluating colony forming unit capacity, expression of MSC markers using flow cytometry, and multidifferential capacity. Secondly, polymerase chain reaction analysis demonstrated that the HAp/DN gel surface significantly enhanced mRNA expression of the eight osteogenic markers (TGF-ß1, BMP-2, Runx2, Col-1, ALP, OPN, BSP, and OCN) in the cultured MSCs at 7 days than the PS surfaces (p < 0.0001), while the DN gel and HAp surfaces provided no or only a slight effect on the expression of these markers except for Runx2. Additionally, the alkaline phosphatase activity was significantly higher in the cells cultured on the HAp/DN gel surface than in the other three material surfaces (p < 0.0001). Thirdly, when the HAp/DN gel plug was implanted into the rabbit bone marrow, MSC marker-positive cells were recruited in the tissue generated around the plug at 3 days, and Runx2 and OCN were highly expressed in these cells. In conclusion, this study demonstrated that the HAp/DN gel surface can differentiate the MSCs into osteogenic cells.

Combination of ultra-purified stem cells with an in situ-forming bioresorbable gel enhances intervertebral disc regeneration.

BACKGROUND: Lumbar intervertebral disc (IVD) herniations are associated with significant disability. Discectomy is the conventional treatment option for IVD herniations but causes a defect in the IVD, which has low self-repair ability, thereby representing a risk of further IVD degeneration. An acellular, bioresorbable, and good manufacturing practice (GMP)-compliant in situ-forming gel, which corrects discectomy-associated IVD defects and prevents further IVD degeneration had been developed. However, this acellular matrix-based strategy has certain limitations, particularly in elderly patients, whose tissues have low self-repair ability. The aim of this study was to investigate the therapeutic efficacy of using a combination of newly-developed, ultra-purified, GMP-compliant, human bone marrow mesenchymal stem cells (rapidly expanding clones; RECs) and the gel for IVD regeneration after discectomy in a sheep model of severe IVD degeneration. METHODS: RECs and nucleus pulposus cells (NPCs) were co-cultured in the gel. In addition, RECs combined with the gel were implanted into IVDs following discectomy in sheep with degenerated IVDs. FINDINGS: Gene expression of NPC markers, growth factors, and extracellular matrix increased significantly in the co-culture compared to that in each mono-culture. The REC and gel combination enhanced IVD regeneration after discectomy (up to 24 weeks) in the severe IVD degeneration sheep model. INTERPRETATION: These findings demonstrate the translational potential of the combination of RECs with an in situ-forming gel for the treatment of herniations in degenerative human IVDs. FUNDING: Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Agency for Medical Research and Development, and the Mochida Pharmaceutical Co., Ltd.

The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance.

How mechanical stress affects physical performance via tendons is not fully understood. Piezo1 is a mechanosensitive ion channel, and E756del PIEZO1 was recently found as a gain-of-function variant that is common in individuals of African descent. We generated tendon-specific knock-in mice using R2482H Piezo1, a mouse gain-of-function variant, and found that they had higher jumping abilities and faster running speeds than wild-type or muscle-specific knock-in mice. These phenotypes were associated with enhanced tendon anabolism via an increase in tendon-specific transcription factors, Mohawk and Scleraxis, but there was no evidence of changes in muscle. Biomechanical analysis showed that the tendons of R2482H Piezo1 mice were more compliant and stored more elastic energy, consistent with the enhancement of jumping ability. These phenotypes were replicated in mice with tendon-specific R2482H Piezo1 replacement after tendon maturation, indicating that PIEZO1 could be a target for promoting physical performance by enhancing function in mature tendon. The frequency of E756del PIEZO1 was higher in sprinters than in population-matched nonathletic controls in a small Jamaican cohort, suggesting a similar function in humans. Together, this human and mouse genetic and physiological evidence revealed a critical function of tendons in physical performance, which is tightly and robustly regulated by PIEZO1 in tenocytes.

Force-triggered rapid microstructure growth on hydrogel surface for on-demand functions.

Living organisms share the ability to grow various microstructures on their surface to achieve functions. Here we present a force stamp method to grow microstructures on the surface of hydrogels based on a force-triggered polymerisation mechanism of double-network hydrogels. This method allows fast spatial modulation of the morphology and chemistry of the hydrogel surface within seconds for on-demand functions. We demonstrate the oriented growth of cells and directional transportation of water droplets on the engineered hydrogel surfaces. This force-triggered method to chemically engineer the hydrogel surfaces provides a new tool in addition to the conventional methods using light or heat, and will promote the wide application of hydrogels in various fields.

Influence of the charge relay effect on the silanol condensation reaction as a model for silica biomineralization.

The catalytic effect of various sequential peptides for silica biomineralization has been studied. In peptide sequence design, lysine (K) and histidine (H) were selected as the standard amino acids and aspartic acid (D) was selected to promote the charge relay effects, such as in the enzyme active site. Therefore, homopolypeptides (K(10) and H(10)), block polypeptides (K(5)D(5) and H(5)D(5)), and alternate polypeptides [(KD)(5) and (HD)(5)] were designed, and the dehydration reaction ability of trimethylethoxysilane was investigated as a quantitative model of silica mineralization. The catalytic activity per basic residue of alternate polypeptide was the highest because of the charge relay effects at the surface of the peptide. In silica mineralization using tetraethoxysilane, spherical silica particles were obtained, and their size is related to the catalytic activities of the peptides in the model systems. From these results, the effect of the functional group combination by the peptide sequence design enables the control of the efficiency of mineralization and preparation of specific inorganic materials.

Multistep growth mechanism of calcium phosphate in the earliest stage of morphology-controlled biomineralization.

We studied the effect of surface-functional-group position on precipitate morphology in the earliest stage of calcium phosphate biomineralization and determined the detailed mechanism of precipitation starting from nucleation to precipitate growth. The biomineralization template was a ß-sheet peptide scaffold prepared by adsorption with carboxyl groups arranged at strict 7 Å intervals. Phosphate was then introduced. Within 10 s, highly ordered embryos of calcium phosphate were formed and confined by a peptide nanofiber pattern. They repeatedly nucleated and dissolved, with the larger embryos absorbing the smaller ones in a clear demonstration of an Ostwald-ripening-like phenomenon, then aggregated in a line pattern, and finally formed highly ordered nanofibers of amorphous calcium phosphate. This multistep growth process constitutes the earliest stage of biomineralization.

Tough Double Network Hydrogel and Its Biomedical Applications.

Soft and wet hydrogels have many similarities to biological tissues, though their mechanical fragility had been one of the biggest obstacles in biomedical applications. Studies and developments in double network (DN) hydrogels have elucidated how to create tough gels universally based on sacrificial bond principles and opened a path for biomedical application of hydrogels in regenerative medicine and artificial soft connective tissues, such as cartilage, tendon, and ligament, which endure high tension and compression. This review explores a universal toughening mechanism for and biomedical studies of DN hydrogels. Moreover, because the term sacrificial bonds has been mentioned often in studies of bone tissues, consisting of biomacromolecules and biominerals, recent studies of gel-biomineral composites to understand early-stage osteogenesis and to simulate bony sacrificial bonds are also summarized.

Bone Marrow Aspirate Concentrate Combined with in Situ Forming Bioresorbable Gel Enhances Intervertebral Disc Regeneration in Rabbits.

BACKGROUND: The current surgical procedure of choice for intervertebral disc (IVD) herniation is discectomy, which induces postoperative IVD degeneration. Thus, cell-based therapies, as a 1-step simple procedure, are desired because of the poor capacity of IVDs for self-repair. The aim of this study was to investigate the repair efficacy of ultra-purified alginate (UPAL) gels containing bone marrow aspirate concentrate (BMAC) for the treatment of discectomy-associated IVD degeneration in rabbits. METHODS: The mechanical properties of 3 types of gels-UPAL, UPAL containing bone marrow-derived mesenchymal stem cells (BMSCs), and UPAL containing BMAC-were evaluated. Forty rabbits were assigned to 5 groups: intact control, discectomy (to make the cavity), UPAL (implantation of the UPAL gel after discectomy), BMSCs-UPAL (implantation of a combination of autogenic BMSCs and UPAL gel after discectomy), and BMAC-UPAL (implantation of a combination of BMAC and UPAL gel after discectomy). The gels were implanted at 4 weeks after induction of IVD degeneration. At 4 and 12 weeks, magnetic resonance imaging (MRI) as well as histological and immunohistochemical analyses were performed to analyze IVD degeneration qualitatively and the viability of the implanted cells. RESULTS: There was no significant difference among the 3 types of gels in terms of the results of unconfined compression tests. The implanted cells survived for 12 weeks. The histological grades of the BMSCs-UPAL (mean and standard deviation, 2.50 ± 0.53; p < 0.001) and BMAC-UPAL (2.75 ± 0.64, p = 0.001) showed them to be more effective in preventing degeneration than UPAL gel alone (3.63 ± 0.52). The effectiveness of BMAC-UPAL was not significantly different from that of BMSCs-UPAL, except with respect to type-II collagen synthesis. CONCLUSIONS: BMAC-UPAL significantly enhanced the repair of IVD defects created by discectomy. This approach could be an effective therapeutic strategy owing to its simplicity and cost-effectiveness compared with cell therapy using culture-expanded BMSCs. CLINICAL RELEVANCE: Local administration of the BMAC combined with UPAL gel could be an effective therapeutic strategy to enhance IVD repair after discectomy.