Human corneal endothelial cell transplantation with nanocomposite gel sheet preserves corneal stability in post-corneal transplant bullous keratopathy: a 16-year follow-up.

Post-corneal transplantation endothelial decompensation and subsequent bullous keratopathy often result in unfavorable clinical outcomes regardless of the treatment strategy employed. In this report, we present the outcomes of a patient managed with in vitro expanded human corneal endothelial cell (HCEC) transplantation facilitated by a nanocomposite gel (NC gel) sheet over 16 years. A 40-year-old male patient who presented with signs of graft failure after penetrating keratoplasty underwent HCEC transplantation. Additionally, HCECs were obtained from a deceased donor, cultured in vitro, and transplanted onto an NC gel sheet as a temporary scaffold to support the transplanted cells until engraftment. At the 16-year follow-up, the cornea had remained stable and did not exhibit active disease manifestations. Notably, no new bullae were formed, and the epithelial surface appeared smooth without signs of active fluid transport abnormalities. Although a slight reduction in corneal thickness was observed, the disease-free region at the time of the intervention remained transparent. HCEC transplantation with NC gel sheets is a promising, minimally invasive approach for achieving long-term corneal stability in cases of bullous keratopathy following corneal graft failure. Importantly, this technique circumvents the need for complex procedures and utilizes corneal endothelial precursors derived from donor corneas discarded for lack of sufficient endothelial cells. After in vitro culture, these cells were successfully transplanted in three patients, proving that one donated eye can be useful in treating three eyes of three patients. This technique addresses the donor cornea shortage concerns and makes our concept "an-eye-for-eyes", a reality.

Association Behavior of Amphiphilic ABA Triblock Copolymer Composed of Poly(2-methoxyethyl acrylate) (A) and Poly(ethylene oxide) (B) in Aqueous Solution.

Poly(2-methoxyethyl acrylate) (PMEA) and poly(ethylene oxide) (PEO) have protein-antifouling properties and blood compatibility. ABA triblock copolymers (PMEAl-PEO11340-PMEAm (MEOMn; n is average value of l and m)) were prepared using single-electron transfer-living radical polymerization (SET-LRP) using a bifunctional PEO macroinitiator. Two types of MEOMn composed of PMEA blocks with degrees of polymerization (DP = n) of 85 and 777 were prepared using the same PEO macroinitiator. MEOMn formed flower micelles with a hydrophobic PMEA (A) core and hydrophilic PEO (B) loop shells in diluted water with a similar appearance to petals. The hydrodynamic radii of MEOM85 and MEOM777 were 151 and 108 nm, respectively. The PMEA block with a large DP formed a tightly packed core. The aggregation number (Nagg) of the PMEA block in a single flower micelle for MEOM85 and MEOM777 was 156 and 164, respectively, which were estimated using a light scattering technique. The critical micelle concentrations (CMCs) for MEOM85 and MEOM777 were 0.01 and 0.002 g/L, respectively, as determined by the light scattering intensity and fluorescence probe techniques. The size, Nagg, and CMC for MEOM85 and MEOM777 were almost the same independent of hydrophobic DP of the PMEA block.

Instant Strong Adhesive Behavior of Nanocomposite Gels toward Hydrophilic Porous Materials.

We investigated the adhesion behavior of nanocomposite hydrogels (NC gels), consisting of unique organic (polymer)-inorganic (clay) network structures, toward inorganic and organic materials. The NC gels exhibit instant and strong adhesion to inorganic and organic substrates with hydrophilic porous surfaces. The NC gels instantly adhere to hydrophilic porous substrates (e.g., unglazed ceramic surfaces and polymer membranes) through simple light contact. In addition, a small piece of NC gel effectively joined two substrate samples (e.g., concrete blocks and bricks) through lamination of the interposing NC gel. The resulting conjoined materials were unable to be separated at the gel-substrate interface; rather, the gel itself fractured upon separation, which indicates that the adhesive strength at the interface is greater than the tensile strength of the NC gel. With the exception of NC gels with very high clay concentrations ( Cclay's), instant strong adhesion and cohesive failure by subsequent stretching were observed for almost all NC gels composed of different polymers or different Cclay values. A thermoresponsive NC gel was reversibly adhered and could be peeled from the surface by stretching (adhesive failure) at a temperature above its transition temperature. The mechanism of instant strong adhesion or reversible adhesion is discussed based on dangling chains that exist on the surfaces of the NC gels composed of polymer-clay networks. The cut surface of an NC gel generally exhibited a higher adhesive strength than the as-prepared surface because of longer dangling chains.

Human corneal endothelial cell transplantation using nanocomposite gel sheet in bullous keratopathy.

Transplantation of in vitro expanded human corneal endothelial precursors (HCEP) cells using a nanocomposite (D25-NC) gel sheet as supporting material in bovine's cornea has been earlier reported. Herein we report the transplantation of HCEP cells derived from a cadaver donor cornea to three patients using the NC gel sheet. In three patients with bullous keratopathy, one after cataract surgery, one after trauma and another in the corneal graft, earlier performed for congenital corneal dystrophy, not amenable to medical management HCEP cells isolated from a human cadaver donor cornea in vitro expanded using a thermoreversible gelation polymer (TGP) for 26 days were divided into three equal portions and 1.6 × 105 HCEP cells were injected on to the endothelium of the affected eye in each patient using the D25-NC gel sheet as a supporting material. The sheets were removed after three days. The bullae in the cornea disappeared by the 3rd-11th post-operative day in all the three patients. Visual acuity improved from Perception of light (PL)+/Projection of rays (PR)+ to Hand movements (HM)+ in one of the patients by post-operative day 3 which was maintained at 18 months follow-up. At 18 months follow-up, in another patient the visual acuity had improved from HM+ to 6/60 while in the third patient, visual acuity remained HM+ as it was prior to HCEP transplantation. There were no adverse effects during the follow-up in any of the patients.

Reversible generation of large retractive tensile forces in isometric chemo-mechanical actuators composed of nanocomposite hydrogels and aqueous NaCl solutions.

This article addresses the generation of large mechanical forces in muscle-like isometric actuator systems composed of stimuli-responsive nanocomposite hydrogels (NC gels) and aqueous NaCl solutions. NC gels with poly(N-isopropylacrylamide)-clay (PNIPA-clay) network structures exhibit high mechanical toughnesses and reversible swelling/deswelling behavior in the absence/presence of NaCl. NC gels constrained to constant lengths in brine generate enhanced contractive forces due to the salt-induced coil-to-globule transitions that are more than ten times larger than those induced thermally. A retractive tensile force (4 N/170 kPa), comparable to that of human muscle, was repeatedly generated at 20 °C using an appropriately composed NC gel (clay: 2 mol%, PNIPA: 2 M), and by alternating the NaCl concentration between 0 and 5 M. This phenomenon is attributed to the combined effects of enhanced deswelling behavior resulting from the salt-induced coil-to-globule transition of PNIPA and the high stiffness of the deswollen gel due to the NaCl-strengthened PNIPA-clay network.

Clay-Alcohol-Water Dispersions: Anomalous Viscosity Changes Due to Network Formation of Clay Nanosheets Induced by Alcohol Clustering.

Clay-alcohol-water ternary dispersions were compared with alcohol-water binary mixtures in terms of viscosity and optical absorbance. Aqueous clay dispersions to which lower alcohols (ethanol, 1-propanol, 2-propanol, and tert-butanol) were added exhibited significant viscosity anomalies (maxima) when the alcohol content was 30-55 wt %, as well as optical absorbance anomalies (maxima). The maximum viscosity (ηmax) depended strongly on the clay content and varied between 300 and 8000 mPa·s, making it remarkably high compared with the viscosity anomalies (2 mPa·s) observed in alcohol-water binary mixtures. The alcohol content at ηmax decreased as the hydrophobicity of the alcohol increased. The ternary dispersions with viscosity anomalies exhibited thixotropic behaviors. The effects of other hydrophilic solvents (glycols) and other kinds of clays were also clarified. Based on these findings and the average particle size changes, the viscosity anomalies in the ternary dispersions were explained by alcohol-clustering-induced network formation of the clay nanosheets. It was estimated that 0.9, 1.7, and 2.5 H2O molecules per alcohol molecule were required to stabilize the ethanol, 2-propanol, and tert-butanol, respectively, in the clay-alcohol-water dispersions.

Antithrombogenic Properties of Amphiphilic Block Copolymer Coatings: Evaluation of Hemocompatibility Using Whole Blood.

Antithrombogenicity is one of the most critical properties required for materials used in biomedical devices, particularly in devices that contact blood. The antithrombogenicity of surfaces coated with amphiphilic block copolymers composed of hydrophobic poly(2-methoxyethyl acrylate) (M) and hydrophilic poly(N,N-dimethylacrylamide) (D) segments was investigated using plasma protein and whole blood with regard to protein adsorption, thrombus formation, platelet activation, and clotting kinetics. Three types of block copolymers and a random copolymer were synthesized using one-pot reversible addition-fragmentation chain-transfer (RAFT) polymerization under conditions of high yield and high molecular weight. Triblock and 4-arm block copolymers with MDM and (MD)4 architecture, respectively, showed good adhesion to both organic and inorganic substrates, including polyvinyl chloride (PVC) tubes, and the resulting coated surfaces showed superior protein repellency and hemocompatibility compared to the diblock or random copolymer coatings and noncoated control. In a Chandler-loop method with whole blood, PVC tubes coated with MDM and (MD)4 showed improved thromboresistance and adsorption resistance to blood-derived proteins. This high hemocompatibility was also confirmed with human whole blood by thrombelastography (suppression of blood-clotting behavior in both intrinsic and extrinsic coagulation pathways) and platelet function analyses (significant reductions in the aggregation activity of platelets under two types of stimulation). The antithrombogenicity has been discussed based on the structural analyses of the MDM-coated surface. The results of this study will enable the development of more effective biomedical and analytical devices with excellent antithrombogenic characteristics by using a simple and environmentally friendly approach.

Clay-supported novel bimetallic core-shell Co-Pt and Ni-Pt nanocrystals with high catalytic activities.

We demonstrate the striking potential of exfoliated clay (synthetic hectorite; Laponite XLG) platelets to prepare bimetallic (Co-Pt and Ni-Pt) NCs with well-defined structures. Catalytic studies show a strong bimetallic synergistic effect of the core-shell NCs; their catalytic activities are much higher than those of monometallic NCs and other bimetallic core-shell NCs.

Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

Novel bimetallic core-shell nanocrystal-clay composites with superior catalytic activities.

Clay (synthetic hectorite; Laponite XLG) plays a very crucial role in the formation and stabilization of core-shell nanocrystals and affords high stability, large BET surface area and stimulates the exceptional catalytic activity of the core-shell NCs.

Preparation and characterization of highly planar flexible silver crystal belts.

We report a novel simple one-pot strategy for fabricating pure and highly planar silver (Ag) crystal belts. Unique single-crystal Ag belts (high width-to-thickness ratio ~50) were successfully synthesized in high yield (80 wt%) by reducing AgNO3 using N,N,N',N'-tetramethylethylenediamine (TEMED) as a reducing and a structure-determining agent in the presence of polyethylene glycol (PEG) under mild conditions.

Successful transplantation of in vitro expanded human cadaver corneal endothelial precursor cells on to a cadaver bovine's eye using a nanocomposite gel sheet.

PURPOSE: In vitro expansion of human corneal endothelial precursor (HCEP) cells has been reported via production of cell aggregated spheres. However, to translate this procedure in human patients warrants maintaining the position of the eyeballs facing down for 36 h, which is not feasible. In this study, we report a method using a nanocomposite (NC) gel sheet to accomplish the integration of HCEP cells to the endothelium of cadaver bovine's eyes. MATERIALS AND METHODS: HCEP cells were isolated from the corneal endothelium of a cadaver human eye and then expanded using a thermoreversible gelation polymer (TGP) as reported earlier. For the study, three cadaver bovine eyes were used. The NC gel sheets were inserted into the bovine eyes', aligned and suture-fixed in position under the host endothelium. HCEP cells previously expanded in the TGP were harvested and injected using a 26-gauge syringe between the endothelium and the NC gel sheet. The eyes were left undisturbed for three hours following which the NC gel sheets were gently removed. The corneas were harvested and subjected to histopathological studies. RESULTS: Histopathological studies showed that all the three corneas used for NC gel sheet implantation showed the presence of engrafted HCEP cells, seen as multi-layered cells over the native endothelium of the bovine cornea. Examination of the NC gel sheets used for implantation showed that only very few corneal endothelial cells remained on the sheets amounting to what could be considered negligible. CONCLUSION: The use of the NC gel sheet makes HCEP cell transplantation feasible for human patients. Further in vitro basic studies followed by translational studies are necessary to bring this method for clinical application in appropriate indications.

Superior CO catalytic oxidation on novel Pt/clay nanocomposites.

Nanostructured novel Pt/Clay nanocomposites consisting of well-defined Pt nanoparticles prepared by clay-mediated in situ reduction displays very high thermal stability, large BET surface area and superior catalytic activity for CO oxidation as compared to a model reference Pt/SiO2 catalysts. CO oxidation has attracted renewed attention because of its technological importance in the area of pollution control. The Pt/Clay system consisting of Pt nanoparticles strongly immobilized between the atomic layers of clay inhibits nanoparticle sintering and loss of catalytic activity even after prolonged heating at high temperatures. At elevated temperatures (300 °C), the Pt/Clay system demonstrates significant enhancement of catalytic activity, with almost 100% CO conversion in less than 5 min. Emphasis is given to the role played by the clay supporting material which is chemically and thermally stable under the catalytic conditions of exhaust purification.

Super-thermostable platinum nanoparticles on fluorinated clay.

Pt/fluorinated clay nanocomposites were prepared by clay-mediated in situ reduction; these nanocomposites demonstrated ultrahigh Pt nanoparticle thermal stability (up to 1000 °C), large BET surface area (379 m(2) g(-1)), uniform dispersion in aqueous media, and intriguing catalytic properties.

Synthesis of highly active and thermally stable nanostructured Pt/clay materials by clay-mediated in situ reduction.

Novel and intriguing one-pot in situ method for the preparation of nanostructured Pt-clay materials under simple conditions is reported. In this synthesis, an inorganic clay mineral such as synthetic hectorite ("Laponite XLG") or natural montmorillonite ("Kunipia F") serves as a mild and effective reducing agent for Pt ions, which is uncommon for such a clay system, and also acts as an outstanding stabilizer for the resulting Pt nanoparticles. In aqueous solution, exfoliated colloidal clay platelets forms complex with Pt ions in the initial stage of mixing. Devoid of any organic dispersants or external reducing agents, subsequently, the Pt nanoparticles (3-6 nm) generated by clay-assisted in situ reduction of Pt ions successfully anchored onto the clay nanoplatelets. The Pt-clay material features a very high surface area (312 m(2) g(-1)) and has excellent catalytic activity, as was kinetically evaluated via the reduction of 4-nitrophenol with NaBH(4). After drying, this remarkably stable nanocomposite is completely redispersible in water and displays extreme thermal stability (up to 500 °C). On the basis of these results, this synthetic strategy is anticipated to be a very simple, economical, and green approach for the synthesis of nanostructured Pt-clay materials.

Proliferation and harvest of human mesenchymal stem cells using new thermoresponsive nanocomposite gels.

For tissue engineering and regenerative medicine, stem cells should be effectively cultured in vitro. New thermoresponsive nanocomposite gels (MD-NC gels), consisting of inorganic clay (hectorite) and copolymers composed of hydrophobic 2-methoxyethyl acrylate (MEA) and hydrophilic N,N-dimethylacrylamide (DMAA) units, could be applied in cell culture and cell harvesting without trypsinization, specifically using mesenchymal stem cells (MSCs). The composition of the MD-NC gel (the ratio of the two monomer types and the clay content) was found to determine its swelling properties in the culture medium, thermosensitivity, protein adsorption, and cell attachment and proliferation. Various kinds of human cells, including MSCs, osteoblast (HOS) cells, fibroblast (NHDF) cells, and epithelial cells could be effectively cultured on MD-NC gels. In particular, on an MD10-NC2 gel with relatively low DMAA and clay content, the cells could be harvested by decreasing the temperature, either as a cell sheet (MSCs or NHDF cells) or as a population of suspension cells (HOS cells). Further, it was found that the MD10-NC2 gel is suitable for stem cell differentiation. Because of their thermosensitivity, controllable modulus, and surface properties, MD-NC gels are promising cell culture substrates useful for tissue engineering and regenerative medicine.

Effects of counter ions of clay platelets on the swelling behavior of nanocomposite gels.

The effects of replacing the native Na(+) counter ions associated with the clay platelets by various other cations on the swelling behavior of nanocomposite (NC) gels consisting of an organic (polymer)/inorganic (clay) network were investigated. The negative surface charge of the clay platelet conferred an ionic nature on the NC gels making them a type of polyelectrolyte gel; consequently, the swelling behavior of the NC gels was strongly influenced by the valence of the co-existing counter ions. NC gels containing monovalent cations such as Na(+), K(+) and Li(+) exhibited large swellings and subsequent deswelling in water after attaining maximum degrees of swelling. In contrast, introduction of multivalent cations such as Ca(2+), Mg(2+), and Al(3+) into NC gels depressed markedly both the swelling and subsequent deswelling. The decreased swelling and suppressed deswelling with multivalent ions were strongly influenced by the initial gel state and result from the formation of additional cross-links through ionic interactions between the clay platelets and the multivalent cations. Also, the similar swelling behaviors were observed for all NC gels with different clay concentration. Further, reversible absorption/desorption and selective absorption of multivalent cations were observed for the NC gels examined.

Self-healing in nanocomposite hydrogels.

Polymer hydrogels with characteristics distinct from those of solid materials are one of the most promising candidates for smart materials. Here, we report that a nanocomposite hydrogel (NC gel) consisting of a unique polymer/clay network structure, can exhibit complete self-healing through autonomic reconstruction of crosslinks across a damaged interface. Mechanical damage in NC gels can be repaired without the use of a healing agent, and even sections of NC gels separated by cutting, from whichever the same or different kinds of NC gel, perfectly (re-)combine by just contacting the cut surfaces together at mildly elevated temperatures. In NC gels, the autonomic fusion of cut surfaces as well as the self-healing could be achieved not only immediately after being cut but also after a long waiting time.

Thermoresponsible cell adhesion/detachment on transparent nanocomposite films consisting of poly(2-methoxyethyl acrylate) and clay.

Soft, transparent and mechanically tough nanocomposite (M-NC) films composed of poly(2-methoxyethyl acrylate) (PMEA) and inorganic clay (hectorite) were studied as substrates for a living cell harvest system. It was found that five cell types could all be cultivated to confluence on the surface of M-NC n films with clay content (n = 10-23 wt%), although the cells hardly cultivated on the surface of chemically-cross-linked PMEA and linear PMEA films. Further, it was found that the cells cultured on the surfaces of M-NC films can be detached, without any enzymatic digestion, by decreasing the medium temperature and/or simultaneously using gentle pipetting. The detached cell was obtained as a single cell or a contiguous cell sheet, both of which were viable and recultured. From the compositions and surface properties, it was estimated that the cell culture and subsequent cell detachment were attributed to the unique PMEA/clay network. The new soft nanocomposite is potentially a very promising substrate for tissue engineering.

Molecular Characteristics of Poly(N-isopropylacrylamide) Separated from Nanocomposite Gels by Removal of Clay from the Polymer/Clay Network.

The extraordinary mechanical and swelling/deswelling properties of nanocomposite (NC) gels are attributed to their unique organic (polymer)/inorganic (clay) network structure. In this study, poly(N-isopropylacrylamide) (PNIPA) was successfully separated from an NC gel network by decomposing the clay (hectorite) using hydrofluoric acid (HF). A very low HF concentration (0.2 wt.-%) was adequate for the decomposition of the clay without causing any damage to PNIPA. The separated PNIPA had a high $\overline M _{\rm w}$ (=5.5 × 10(6) g · mol(-1) ). Also, $\overline M _{\rm w}$ was almost constant regardless of the clay concentration (C(clay) = 1-25 × 10(-2) mol · l(-1) ), even though the properties of the NC gel varied widely over this C(clay) range. Comparisons of NC gels, PNIPA, and SiO(2) -NC gels indicated that the clay platelets specifically play an important role in NC gels.