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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.

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.

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A previous study reported that intercellular adhesion molecule-1 (ICAM-1) expression by human vascular endothelial cells (HUVEC) is augmented by intracellular signal transmission mainly through the protein kinase C (PKC) system stimulated by TXA2 receptors. In the present study, we show that a TXA2 receptor agonist, U46619, augments the expression of not only ICAM-1, but also vascular cell adhesion molecule-1 (VCAM-1) or endothelial leucocyte adhesion molecule-1 (ELAM-1) in HUVEC both at protein and mRNA levels. Pretreatment with SQ29,548 (a TXA2 receptor antagonist) or PKC inhibitors greatly diminished the extent of U46619-induced mRNA accumulation and surface expression of the adhesion molecules. An inhibitor of nuclear factor kappaB (NF-kappaB) activation, PDTC, diminishes U46619-induced VCAM-1 mRNA accumulation. NAC, which inhibits NF-kappaB and activation protein 1 (AP-1) binding activity, inhibits the expression of ICAM-1 or ELAM-1 at protein and mRNA levels. These findings suggest that ICAM-1 or ELAM-1 expression of HUVEC stimulated via TXA2 receptors is augmented by induction of NF-kappaB and AP-1 binding activity through the PKC system, and that VCAM-1 expression is augmented by induction of NF-kappaB binding activity.