Takemi Matsui

Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations.

PHOTOCROSSLINKABLE CHITOSAN HYDROGEL CONTAINING FIBROBLAST GROWTH FACTOR-2 STIMULATES WOUND HEALING IN HEALING-IMPAIRED DB/DB MICE

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) resulted within 30s in an insoluble, flexible hydrogel. About 20% of the FGF-2molecules were released from the FGF-2-incorporated chitosan hydrogel into phosphate buffered saline (PBS) within 1 day, after which no further significant release occurred under in vitro non-degradation conditions of the hydrogel. The FGF-2molecules retained in the chitosan hydrogel remained biologically active, and were released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In order to evaluate its accelerating effect on wound healing, full thickness skin incisions were made on the back of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure in both db/db and db/+ mice. However, the addition of FGF-2 in the chitosan hydrogel further accelerated wound closure in db/db mice, although not in db/+ mice. Histological examination also has demonstrated an advanced granulation tissue formation, capillary formation and epithelialization in wounds treated with FGF-2-incorporated chitosan hydrogels in db/db mice.

Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source.

Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivowas examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs.

Vascularization in vivo caused by the controlled release of fibroblast growth factor-2 from an injectable chitosan/non-anticoagulant heparin hydrogel

Addition of various heparinoids to the lactose-introduced, water-soluble chitosan (CH-LA) aqueous solution produces an injectable chitosan/heparinoid hydrogel. In the present work, we examined the capability of the chitosan/non-anticoagulant heparin (periodate-oxidized (IO(4)-) heparin) hydrogel to immobilize fibroblast growth factor (FGF)-2, as well as the controlled release of FGF-2 molecules from the hydrogel in vitro and in vivo. The hydrogel was biodegraded in about 20 days after subcutaneous injection into the back of a mouse. When the FGF-2-incorporated hydrogel was subcutaneously injected into the back of both mice and rats, a significant neovascularization and fibrous tissue formation were induced near the injected site. These results indicate that the controlled release of biologically active FGF-2 molecules is caused by biodegradation of the hydrogel, and that subsequent induction of the vascularization occurs.

Acceleration of wound contraction and healing with a photocrosslinkable chitosan hydrogel

Application of ultraviolet light irradiation to a photocrosslinkable chitosan aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 seconds. In order to evaluate its accelerating effect on wound healing, full‐thickness skin incisions were made on the backs of mice and subsequently a photocrosslinkable chitosan aqueous solution was added into the wound and irradiated with UV light for 90 seconds. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing compared with the untreated controls. Histological examination also showed an advanced contraction rate on the first 2 days and tissue fill rate on days 2 to 4 in the chitosan hydrogel‐treated wounds. Furthermore, in cell culture studies, chitosan hydrogel culture medium supplemented with 5% fetal–bovine serum was found to be chemoattractant for human dermal fibroblasts in an invasion chamber assay using filters coated with Matrigel and in a cell migration assay. Due to its ability to accelerate wound contraction and healing, chitosan hydrogel may become accepted as an occlusive dressing for wound management.

Acceleration of wound healing in healing-impaired db/db mice with a photocrosslinkable chitosan hydrogel containing fibroblast growth factor-2.

Application of ultraviolet light irradiation to a photocrosslinkable chitosan (Az‐CH‐LA) aqueous solution including fibroblast growth factor‐2 (FGF‐2) results within 30 seconds in an insoluble, flexible hydrogel. The FGF‐2 molecules retained in the chitosan hydrogel remain biologically active and are released from the chitosan hydrogel upon in vivo biodegradation of the hydrogel. To evaluate the accelerating effect on wound healing of this hydrogel, full‐thickness skin incisions were made in the backs of healing‐impaired diabetic (db/db) mice and their normal (db/+) littermates. The mice were later killed, and histological sections of the wound were prepared. The degree of wound healing was evaluated using several histological parameters such as the rate of contraction, epithelialization, and tissue filling. Application of the chitosan hydrogel significantly advanced the rate of contraction on Days 0 to 2 in db/db and db/+ mice. Although the addition of FGF‐2 into the chitosan hydrogel in db/+ mice had little effect, application of the chitosan hydrogel–containing FGF‐2 further accelerated the adjusted tissue filling rate (Days 2 to 4 and Days 4 to 8) in db/db mice. Furthermore, the chitosan hydrogel–containing FGF‐2 markedly increased the number of CD‐34‐positive vessels in the wound areas of db/db mice on Day 4. Thus, the application of chitosan hydrogel–containing FGF‐2 onto a healing‐impaired wound induces significant wound contraction and accelerates wound closure and healing.

Antiviral activity of silver nanoparticle/chitosan composites against H1N1 influenza A virus

Silver nanoparticle (Ag NP)/chitosan (Ch) composites with antiviral activity against H1N1 influenza A virus were prepared. The Ag NP/Ch composites were obtained as yellow or brown floc-like powders following reaction at room temperature in aqueous medium. Ag NPs (3.5, 6.5, and 12.9 nm average diameters) were embedded into the chitosan matrix without aggregation or size alternation. The antiviral activity of the Ag NP/Ch composites was evaluated by comparing the TCID50 ratio of viral suspensions treated with the composites to untreated suspensions. For all sizes of Ag NPs tested, antiviral activity against H1N1 influenza A virus increased as the concentration of Ag NPs increased; chitosan alone exhibited no antiviral activity. Size dependence of the Ag NPs on antiviral activity was also observed: antiviral activity was generally stronger with smaller Ag NPs in the composites. These results indicate that Ag NP/Ch composites interacting with viruses exhibit antiviral activity.

Preparation and characterization of low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as FGF-2 carrier

We produced low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as a carrier for heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2). A mixture of low-molecular-weight heparin (MW: about 5000 Da, 6.4 mg/mL) and protamine (MW: about 3000 Da, 10 mg/mL) at a ratio of 7:3 (vol:vol) yields a dispersion of microparticles (1–6 μm in diameter). In this study, diluted low-molecular-weight heparin solution in saline (0.32 mg/mL) mixed with diluted protamine (0.5 mg/mL) at a ratio at 7:3 (vol:vol) resulted in soluble nanoparticles (112.5 ± 46.1 nm in diameter). The generated NPs could be then stabilized by adding 2 mg/mL dextran (MW: 178–217 kDa) and remained soluble after lyophilization of dialyzed LMW-H/P NP solution. We then evaluated the capacity of LMW-H/P NPs to protect activity of FGF-2. Interaction between FGF-2 and LMW-H/P NPs substantially prolonged the biological half-life of FGF-2. Furthermore, FGF-2 molecules were protected from inactivation by heat and proteolysis in the presence of LMW-H/P NPs.

Therapeutic angiogenesis induced by controlled release of fibroblast growth factor‐2 from injectable chitosan/non‐anticoagulant heparin hydrogel in a rat hindlimb ischemia model

The addition of non‐anticoagulant heparin [periodate‐oxidized (IO4) heparin] and fibroblast growth factor (FGF)‐2 to a viscous water‐soluble chitosan (CH‐LA) aqueous solution produces an injectable FGF‐2/CH‐LA/IO4‐heparin hydrogel. The purpose of this study was to examine the ability of the injected FGF‐2/CH‐LA/IO4‐heparin hydrogel to induce vascularization and fibrous tissue formation. FGF‐2/CH‐LA/IO4‐heparin hydrogels (100 μL of hydrogel consisting of 20 mg/mL of CH‐LA, 2 mg/mL of IO4‐heparin, and 50 μg/mL of FGF‐2) were subcutaneously injected into the backs of wound healing‐impaired diabetic (db/db) mice. Furthermore, the effect of percutaneous injection of FGF‐2/CH‐LA/IO4‐heparin hydrogel at eight sites (25 μL/site) into ischemic left lower limbs of rats was examined from day 4 to at least day 28 postinjection. The injection of FGF‐2/CH‐LA/IO4‐heparin hydrogels into the backs of db/db mice resulted in significant increases in blood vessel formation, significant vascularization, and fibrous tissue formation near the injection site. Injection of FGF‐2/CH‐LA/IO4‐heparin hydrogel into ischemic left lower limbs of rats also significantly recovered and increased blood flow and blood oxygen in the calf and thigh. These results indicate that the controlled release of biologically active FGF‐2 molecules from FGF‐2/CH‐LA/IO4‐heparin induces angiogenesis and possibly collateral circulation in db/db mice and the ischemic limbs of rats.

A novel method to prevent secondary exposure of medical and rescue personnel to toxic materials under biochemical hazard conditions using microwave radar and infrared thermography

In order to prevent secondary exposure of medical personnel to toxic materials under biochemical hazard conditions, we performed a noncontact determination of exposure to toxic conditions via 1215-MHz microwave radar and thermography. A toxic condition was induced by intravenous administration of lipopolysaccharide (LPS) in rabbits. The exposure to LPS was determined by linear discriminant analysis using noncontact derived variables.