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A photocrosslinkable chitosan to which both azide and lactose moieties were introduced (Az-CH-LA) was prepared as a biological adhesive for soft tissues and its effectiveness was compared with that of fibrin glue. Introduction of the lactose moieties resulted in a much more water-soluble chitosan at neutral pH. Application of ultraviolet light (UV) irradiation to photocrosslinkable Az-CH-LA produced an insoluble hydrogel within 60 s. This hydrogel firmly adhered two pieces of sliced ham with each other, depending upon the Az-CH-LA concentration. The binding strength of the chitosan hydrogel prepared from 30-50 mg/mL of Az-CH-LA was similar to that of fibrin glue. Compared to the fibrin glue, the chitosan hydrogel more effectively sealed air leakage from pinholes on isolated small intestine and aorta and from incisions on isolated trachea. Neither Az-CH-LA nor its hydrogel showed any cytotoxicity in cell culture tests of human skin fibroblasts, coronary endothelial cells, and smooth muscle cells. Furthermore, all mice studied survived for at least 1 month after implantation of 200 microL of photocrosslinked chitosan gel and intraperitoneal administration of up to 1 mL of 30 mg/mL of Az-CH-LA solution. These results suggest that the photocrosslinkable chitosan developed here has the potential of serving as a new tissue adhesive in medical use.

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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.

Water-insoluble fragmin/protamine microparticles of about 0.5-1 mum in diameter were prepared by simple mixing of low-molecular-weight heparin (fragmin) with protamine. We investigated the capability of these microparticles to immobilize fibroblast growth factor (FGF)-2, to protect FGF-2 against degradation, to enhance FGF-2 activity, and to facilitate controlled release of FGF-2. FGF-2 bound to the fragmin/protamine microparticles with high affinity (Kd = 2.08 x 10(-9) M) and the half-life of FGF-2-activity was prolonged substantially through binding of FGF-2 to the microparticles, by protection of FGF-2 from inactivation by heat and proteolysis. After subcutaneous injection into the back of mice, the fragmin/protamine microparticles underwent biodegradation and disappeared in about 2 weeks. A similar injection of FGF-2-containing microparticles resulted in significant neovascularization and fibrous tissue formation near the injection site after 1 week. These results indicate that controlled release of biologically active FGF-2 occurs through both slow diffusion and biodegradation of the microparticles, with subsequent induction of neovascularization. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009.

Application of ultraviolet (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted within 10 s in an insoluble, flexible hydrogel. A low molecular weight acidic molecule like trypan blue and various high molecular weight molecules such as bovine serum albumin (BSA), heparin and protamine were all retained within the hydrogel, while a low molecular weight basic molecule like toluidine blue was rapidly released from the hydrogel. In the present work, we examined the retaining capability of the chitosan hydrogel for growth factors and controlled release of growth factors from the chitosan hydrogel in vitro and in vivo. Fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor(165) (VEGF(165)), heparin-binding epidermal growth factor (HB-EGF) in phosphate buffered saline (PBS) were mixed with Az-CH-LA aqueous solution to form growth factor-incorporated chitosan hydrogels. About 10-25% of the growth factor was released from a growth factor-incorporated chitosan hydrogel into PBS within the first day, after which no further substantial release took place. The growth factors interacted with Az-CH-LA molecules poly-ion complexation, and probably were unable to be released after the first day under the in vitro nondegradation conditions of the hydrogel. Although the FGF-1, FGF-2, and VEGF(165)-incorporated chitosan hydrogels on a culture plate significantly stimulated HUVEC growth, the stimulating activity of the growth factor-incorporated chitosan hydrogel was completely cancelled out by washing the hydrogel with PBS solution for 3 days or more. The stimulating activity on the HUVEC growth were however highly recovered by treating the washed growth factor-incorporated chitosan hydrogel during 7 days with chitinase and chitosanase to partly degrade the hydrogel, strongly suggesting that the growth factors within the hydrogel retained their biologically active forms. The chitosan hydrogel (100 microl) when implanted into the back of a mouse was biodegraded in about 10-14 days. When FGF-1- and FGF-2-incorporated chitosan hydrogels were subcutaneously implanted into the back of a mouse, significant neovascularization was induced near the implanted site of the FGF-1- and FGF-2-incorporated chitosan hydrogels. Furthermore, addition of heparin with either FGF-1 or FGF-2 into the hydrogel resulted in a significantly enhanced and prolonged vascularization effect. These results indicate that the controlled release of biologically active FGF-1 and FGF-2 with heparin is caused by biodegradation of the chitosan hydrogel, and subsequent induction of vascularization.

Adipose tissue-derived stromal cells (ATSCs) have recently gained widespread attention as a potential alternate source to bone marrow-derived mesenchymal stem cells with a proliferative capacity and a similar ability to undergo multilineage differentiation. In this study, we evaluated the effectiveness of freshly isolated autologous ATSCs-containing atelocollagen matrix with silicon membrane (ACMS) on wound healing of diabetic (db/db) mice. Cultured ATSCs from (db/db) mice secreted significant amounts of growth factors and cytokines, which are suitable for wound repair. Two full thickness round skin defects were made on the backs of healing-impaired db/db mice. Freshly isolated autologous ATSCs-containing ACMS or ACMS alone were applied to the wounds. Twelve mice were treated and then killed at 1 or 2 weeks (n = 6 each). Histologic sections of the wounds were prepared at each time period after treatment. Histologic examination demonstrated significantly advanced granulation tissue formation, capillary formation, and epithelialization in diabetic healing-impaired wounds treated with autologous ATSCs-containing ACMS, compared with mice treated with ACMS alone. These results suggested that transplantation of autologous ATSCs-containing ACMS significantly accelerated wound healing in diabetic healing-impaired db/db mice.