Hirofumi Yura

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.

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.

Heparin-carrying polystyrene to mediate cellular attachment and growth via interaction with growth factors.

Various sugar-carrying polystyrenes (PSs), which consist of synthetic styrene and sugar moieties, are glycoconjugates that are able to attach to polymeric surfaces. Heparin-carrying PS (HCPS) is especially able to retain the binding of heparin-binding growth factors (GFs) such as vascular endothelial GF 165 (VEGF(165)) or fibroblast GF 2 (FGF-2). Human skin fibroblast cells, human coronary smooth muscle cells, and human coronary endothelial cells have good adherence to the HCPS-coated plate. The growth rate of fibroblast cells on HCPS-coated plates is higher than or comparable to fibronectin-coated, gelatin-coated, or tissue culture treated plates, and the HCPS coating inhibits the growth of smooth muscle cells. On the other hand, the growth rate of endothelial cells on HCPS-coated plates in the presence of either VEGF(165) or FGF-2 is comparable to that on fibronectin-coated, gelatin-coated, and tissue culture treated plates. Endothelial cells grow at a higher rate on HCPS-coated plates retained with either VEGF(165) or FGF-2 than on the other coated plates. These results indicate that growth of various cells can be controlled by the HCPS coating, thereby retaining the bioactivity of molecules such as heparin-binding GFs. Thus, HCPS-coated surfaces control selective growth of various cells.

Photocrosslinkable chitosan: an effective adhesive with surgical applications

Abstract Background : As an effective adhesive with surgical applications, we designed a chitosan molecule (Az-CH-LA) that can be photocrosslinked by ultraviolet (UV) light irradiation, thus forming a hydrogel. The purpose of this work was to evaluate the effectiveness and safety of the photocrosslinkable chitosan hydrogel as an adhesive with surgical applications. Methods : The sealing ability of chitosan was evaluated as a bursting pressure, using the thoracic aorta, trachea, and lungs of farm pigs and was assessed in a rabbit model. The carotid artery and lung of rabbits were punctured with a needle and the use of the chitosan hydrogel stopped the bleeding and air leakage, respectively. In vivo chitosan degradability and biological responses were assessed histologically. Results : The sealing ability of the chitosan hydrogel was found to be similar or even stronger than that of fibrin. All rabbits whose carotid arteries or lungs were punctured with a needle and then sealed with the chitosan hydrogel stayed alive during a 1-month period, without bleeding or air leakage from the punctured site. Histological examinations demonstrated that, 30 days after application, a fraction of the chitosan hydrogel was phagocytozed by macrophages, partially degraded, and inducing the formation of fibrous tissue around the hydrogel. Conclusion : Photocrosslinkable chitosan showed strong sealing ability and potential use as a new tissue adhesive in surgical applications.

Photocrosslinkable Chitosan Hydrogel Can Prevent Bone Formation in Both Rat Skull and Fibula Bone Defects

UV light irradiation to a photocrosslinkable chitosan (Az-CH-LA) resulted in an insoluble and flexible hydrogel within 30 s. The purpose of this study was to evaluate the ability of the photocrosslinkable chitosan to inhibit bone formation in the bone defects. A 5-mm-diameter defect was made in the rat calvarium, and then photocrosslinkable chitosan was implanted and irradiated with UV for 30 s. Furthermore, a 2-mm defect was made in the fibula of a rat hind leg, and then photocrosslinkable chitosan was implanted and irradiated with UV. Bone formations in the rat skull and fibula defects with photocrosslinkable chitosan hydrogel were significantly prevented for 8 weeks. Thus, the chitosan hydrogel has an inhibitory effect on bone formation.

Selection of hematopoietic stem cells with a combination of galactose-bound vinyl polymer and soybean agglutinin, a galactose-specific lectin.

BACKGROUND: Selection of hematopoietic stem cells can be used to prevent graft‐versus‐host disease (GVHD) after allograft transplantation. The purpose of the study was to examine a novel cell separation system comprising a galactose‐bound vinyl polymer (Gal‐VP) and soybean agglutinin (SBA), a galactose‐specific lectin.

Development of a Novel Emergency Hemostatic Kit for Severe Hemorrhage

Photocrosslinkable chitosan (Az-CH-LA) contains lactose moieties and photoreactive azide groups, and its viscous solution forms an insoluble hydrogel on exposure to UV irradiation. We previously developed an emergency hemostatic kit using the Az-CH-LA solution, calcium alginate, and a UV irradiation apparatus. However, a suitable UV irradiation apparatus is required to effectively convert the Az-CH-LA solution into a hydrogel, and power supply to use the UV irradiation apparatus may not always be available in a disaster area or battlefield. To address this problem, we produced a portable, battery-powered UV irradiation apparatus constituting a novel hemostatic kit for severe hemorrhage. When the hemostatic kit using the UV irradiation apparatus was examined using a rat model of severe hemorrhage, the survival rate increased up to 73%. Hematological values as markers of hemorrhage did not change significantly over the first 3 days. In this study, we describe the characteristics of a portable UV irradiation apparatus and its use in an emergency hemostatic kit prepared using Az-CH-LA and calcium alginate for severe hemorrhage.

Practicability of prenatal testing using lectin-based enrichment of fetal erythroblasts

The aim of this study was to investigate the practicability and efficiency of lectin‐based isolation of fetal erythroblasts for clinical use in non‐invasive prenatal testing.