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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.

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.

Human adipose tissue, obtained by liposuction, was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). The ATSCs, as well as bone marrow-derived mesenchymal stem cells (BMSCs), have the capacity for renewal and the potential to differentiate into multiple lineages of mesenchymal tissues. These cells are capable of forming bone when implanted ectopically in an appropriate scaffold. The aim of this study was to evaluate a beta-tricalcium phosphate (beta-TCP) as a scaffold and to compare the potential of osteogenic differentiation of ATSCs with BMSCs. Both cell types were loaded into beta-TCP disk and cultured in an osteogenic induction medium. Optimal osteogenic differentiation in ATSCs in vitro, as determined by secretion of osteocalcin, scanning electron microscope, and histology, were obtained in the culturing with the beta-TCP disk. Furthermore, bone formation in vivo was examined by using the ATSC- or BMSC-loaded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts and to synthesize bone in beta-TCP disk as have BMSCs.

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.