Taichi Ito

In Situ Cross-linkable Hyaluronan Hydrogels Containing Polymeric Nanoparticles for Preventing Postsurgical Adhesions

Objective:To develop a combined barrier method and drug delivery system (“hybrid system”) for preventing postoperative peritoneal adhesions, which could combine the biocompatibility and ease of application of in situ cross-linkable hydrogels with the controlled release features of polymeric nanoparticles. Methods:Poly(lactic-co-glycolic acid) nanoparticles were dispersed in aldehyde- and hydrazide-modified hyaluronic acids (HA), then combined via a double-barreled syringe. The material was subjected to mechanical testing and was assayed for in vitro cytotoxicity to murine mesothelial cells. Subsequently, it was tested for biocompatibility by intraperitoneal injection in mice. The hybrids effectiveness in preventing postsurgical adhesions was assessed using a rabbit sidewall defect-cecum abrasion model, where it was applied to both injured surfaces. Results:The in situ hybrid gel system formed a flexible and durable hydrogel in less than 10 seconds. It had low in vitro cytotoxicity. In the mouse, the cross-linked HA maintained the polymeric nanoparticles in the peritoneum for 1 week, which we had previously shown would have cleared in less than 2 days, and no animals developed adhesions. Notably, the hybrid gel, even in the absence of encapsulated drug, was highly effective in preventing peritoneal adhesions in the rabbit model employed. Animals treated with the hybrid (n = 8) had no adhesions in 62.5% of cases, and none had adhesions that could only be separated by sharp dissection. In contrast, only 4.2% of untreated animals (n = 24) had no adhesions, and 58.3% developed adhesions requiring sharp dissection. Conclusions:The hybrid cross-linked HA-nanoparticle system described here appears to be a biocompatible and highly effective adhesion barrier, which could also deliver antiadhesion drugs.

In Situ Cross-Linkable Hydrogel of Hyaluronan Produced via Copper-Free Click Chemistry

Injectable hydrogels are useful in biomedical applications. We have synthesized hyaluronic acids chemically modified with azide groups (HA-A) and cyclooctyne groups (HA-C), respectively. Aqueous HA-A and HA-C solutions were mixed using a double-barreled syringe to form a hydrogel via strain-promoted [3 + 2] cycloaddition without any catalyst at physiological conditions. The hydrogel slowly degraded in PBS over 2 weeks, which was accelerated to 9 days by hyaluronidase, while it rapidly degraded in a cell culture media with fetal bovine serum within 4 days. Both HA-A and HA-C showed good biocompatibility with fibroblast cells in vitro. They were administered using the double-barreled syringe into mice subcutaneously and intraperitoneally. Residue of the hydrogel was cleared 21 days after subcutaneous administration, while it was cleared 7 days after intraperitoneal administration. This injectable HA hydrogel is expected to be useful for tissue engineering and drug delivery systems utilizing its orthogonality.

Rapid Proton Conduction through Unfreezable and Bound Water in a Wholly Aromatic Pore-Filling Electrolyte Membrane

We found that protons rapidly conduct through unfreezable and bound water in a pore-filling electrolyte membrane (PF-membrane), although many ions usually conduct through free water contained in polymer electrolytes. PF-membrane is a unique membrane that can suppress the swelling of filled sulfonated poly(arylene ether sulfone) (SPES) because of its rigid polyimide substrate. Based on low-temperature DSC measurements, this strong suppression of swelling resulting from the special structure of the polymer electrolyte results in unfreezable and bound water only; it does not contain any free water. Protons rapidly conduct through this structure. In addition, the activation energy of the proton conduction decreased from 16.3 to 9.1 kJ/mol in proportion to the increase in the ion exchange capacity (IEC) of the filled SPES, unlike the almost constant values of the SPES-cast membranes. This tendency of PF-membrane occurred because of the structure of the membrane, where the concentration of the sulfonic acid groups increased with increase in IEC, which became possible by squeezing free water using the swelling suppression of filled SPES. Without being constrained by the PF-membrane, this unique proton conduction through the structured water and highly concentrated sulfonic acid groups will help to develop future polymer electrolytes, particularly in the fuel cell field where protons need to conduct at various conditions such as temperatures below 0 degrees C, combined high temperature and low humidity, and the presence of fuels.

Preparation of Monodisperse Chitosan Microcapsules with Hollow Structures Using the SPG Membrane Emulsification Technique

We describe herein successful preparations of monodisperse chitosan microcapsules with hollow structures using the SPG membrane emulsification technique. Two preparation procedures were examined in this study. In the first method, monodisperse calcium alginate microspheres were prepared and then coated with unmodified chitosan. Subsequently, tripolyphosphate treatment was conducted to physically cross-link chitosan and solubilize the alginate core at the same time. In the second method, photo-cross-linkable chitosan was coated onto the monodisperse calcium alginate microspheres, followed by UV irradiation to chemically cross-link the chitosan shell and tripolyphosphate treatment to solubilize the core. For both methods, it was determined that the average diameters of the chitosan microcapsules depended on those of the calcium alginate microparticles and that the microcapsules have hollow structures. In addition, the first physical cross-linking method using tripolyphosphate was found to be preferable to obtain the hollow structure, compared with the second method using chemical cross-linking by UV irradiation. This was because of the difference in the resistance to permeation of the solubilized alginate through the chitosan shell layers.

Rapid engineering of endothelial cell-lined vascular-like structures in in situ crosslinkable hydrogels

Fabrication of perfusable vascular networks in vitro is one of the most critical challenges in the advancement of tissue engineering. Because cells consume oxygen and nutrients during the fabrication process, a rapid fabrication approach is necessary to construct cell-dense vital tissues and organs, such as the liver. In this study, we propose a rapid molding process using an in situ crosslinkable hydrogel and electrochemical cell transfer for the fabrication of perfusable vascular structures. The in situ crosslinkable hydrogel was composed of hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified hyaluronic acid (HA-CHO). By simply mixing these two solutions, the gelation occurred in less than 20 s through the formation of a stable hydrazone bond. To rapidly transfer cells from a culture surface to the hydrogel, we utilized a zwitterionic oligopeptide, which forms a self-assembled molecular layer on a gold surface. Human umbilical vein endothelial cells adhering on a gold surface via the oligopeptide layer were transferred to the hydrogel within 5 min, along with electrochemical desorption of the oligopeptides. This approach was applicable to cylindrical needles 200-700 µm in diameter, resulting in the formation of perfusable microchannels where the internal surface was fully enveloped with the transferred endothelial cells. The entire fabrication process was completed within 10 min, including 20 s for the hydrogel crosslinking and 5 min for the electrochemical cell transfer. This rapid fabrication approach may provide a promising strategy to construct perfusable vasculatures in cell-dense tissue constructs and subsequently allow cells to organize complicated and fully vascularized tissues while preventing hypoxic cell injury.

Preparation of uniform-sized hemoglobin–albumin microspheres as oxygen carriers by Shirasu porous glass membrane emulsification technique

We have developed a new type of artificial oxygen carrier composed of bovine hemoglobin (bHb) and bovine serum albumin (BSA) prepared by Shirasu porous glass (SPG) membrane emulsification technique. The resultant emulsion droplets containing 10 wt% bHb and 5-20 wt% BSA were subsequently cross-linked by glutaraldehyde to form the microspheres. Due to the uniform pore structure of SPG membranes, the average diameters of bHb10-BSAm microspheres were successfully controlled at around 5 μm with a coefficient of variation of around 10%. In addition, the biocompatibility of the carriers depended on their oxyhemoglobin percentage regardless of their same size. Finally, the P50 values of these microspheres ranged from 8.08 to 11.60 mmHg, which showed a high oxygen affinity and an oxygen delivery function.

Enhancing Osteogenic Differentiation of MC3T3-E1 Cells by Immobilizing Inorganic Polyphosphate onto Hyaluronic Acid Hydrogel

In tissue engineering, precise control of cues in the microenvironment is essential to stimulate cells to undergo bioactivities such as proliferation, differentiation, and matrix production. However, current approaches are inefficient in providing nondepleting cues. In this study, we have developed a novel bioactive hydrogel (HAX-PolyP) capable of enhancing tissue growth by conjugating inorganic polyphosphate chains onto hyaluronic acid macromers. The immobilized polyphosphates provided constant osteoconductive stimulation to the embedded murine osteoblast precursor cells, resulting in up-regulation of osteogenic marker genes and enhanced levels of ALP activity. The osteoconductive activity was significantly higher when compared to those stimulated with free-floating polyphosphates. Even at very low concentrations, immobilization of polyphosphates onto the scaffold allowed sufficient signaling leading to more effective osteoconduction. These results demonstrate the potential of our novel material as an injectable bioactive scaffold, which can be clinically useful for developing bone grafts and bone regeneration applications.

Development of carboxymethyl cellulose nonwoven sheet as a novel hemostatic agent.

Carboxymethyl cellulose (CMC) is a plant-derived material that has high biocompatibility and water solubility. We developed a CMC nonwoven sheet as a hemostatic agent by carboxymethylating a continuous filament cellulose nonwoven sheet. The CMC nonwoven sheet was able to absorb water and dissolve in it. The rates of absorption and dissolution depended on the degree of carboxymethylation. After dissolving in blood, CMC accelerated clot development (possibly owing to the incorporation of CMC into fibrin fibers) and increased the viscosity of the blood, both of which would contribute to the improved blood clotting of an injured surface. In vivo experiments using a rat tail cutting method showed that a CMC nonwoven sheet shortened the bleeding time of the tail when applied to the cut surface. The hemostatic effect of the CMC nonwoven sheet was almost at the same level as a commercial hemostatic bandage. These results suggest that a CMC nonwoven sheet could be used as a novel sheet-type hemostatic agent.

Biomolecule-recognition gating membrane using biomolecular cross-linking and polymer phase transition.

We present for the first time a biomolecule-recognition gating system that responds to small signals of biomolecules by the cooperation of biorecognition cross-linking and polymer phase transition in nanosized pores. The biomolecule-recognition gating membrane immobilizes the stimuli-responsive polymer, including the biomolecule-recognition receptor, onto the pore surface of a porous membrane. The pore state (open/closed) of this gating membrane depends on the formation of specific biorecognition cross-linking in the pores: a specific biomolecule having multibinding sites can be recognized by several receptors and acts as the cross-linker of the grafted polymer, whereas a nonspecific molecule cannot. The pore state can be distinguished by a volume phase transition of the grafted polymer. In the present study, the principle of the proposed system is demonstrated using poly(N-isopropylacrylamide) as the stimuli-responsive polymer and avidin-biotin as a multibindable biomolecule-specific receptor. As a result of the selective response to the specific biomolecule, a clear permeability change of an order of magnitude was achieved. The principle is versatile and can be applied to many combinations of multibindable analyte-specific receptors, including antibody-antigen and lectin-sugar analogues. The new gating system can find wide application in the bioanalytical field and aid the design of novel biodevices.

A biocompatible calcium salt of hyaluronic acid grafted with polyacrylic acid.

We have synthesized hyaluronic acid (HA) grafted with polyacrylic acid (PAA) via controlled radical polymerization (CRP) in aqueous media. The grafted HA (HA-g-PAA) showed slow degradation by hyaluronidase compared with unmodified HA as a result of the steric hindrance produced by grafted PAA, and PAA was detached by hydrolysis and enzymatic degradation by lipase. It formed an insoluble salt immediately after mixing with Ca(2+) by the binding between grafted PAA and Ca(2+). Both HA-g-PAA and its salt showed good biocompatibility, especially to mesothelial cells in vitro. Finally, they were administered into mice subcutaneously and intraperitoneally. The residue of the material was observed 7 days after subcutaneous administration, while the material was almost cleared from the peritoneum 7 days after intraperitoneal administration with or without Ca(2+). HA-g-PAA is expected to be applicable to medical uses such as drug delivery in the peritoneum and for materials preventing peritoneal adhesion.