Bioshell Calcium Oxide (BiSCaO) Ointment for the Disinfection and Healing of Pseudomonas aeruginosa-Infected Wounds in Hairless Rats.

Bioshell calcium oxide (BiSCaO) possesses deodorizing properties and broad microbicidal activity. This study aimed to investigate the application of BiSCaO ointment for the prevention and treatment of infection in chronic wounds in healing-impaired patients, without delaying wound healing. The bactericidal activities of 0.04, 0.2, 1, and 5 wt% BiSCaO ointment, 3 wt% povidone iodine ointment, and control (ointment only) were compared to evaluate the in vivo disinfection and healing of Pseudomonas aeruginosa-infected wounds in hairless rats. Treatment of the infected wounds with 0.2 wt% BiSCaO ointment daily for 3 days significantly enhanced wound healing and reduced the in vivo bacterial counts compared with povidone iodine ointment and control (no wound cleaning). Although 5 wt% BiSCaO ointment provided the lowest bacterial counts during 3 days' treatment, it delayed wound healing. Histological examinations showed significantly advanced granulation tissue and capillary formation in wounds treated with 0.2 wt% BiSCaO ointment for 3 days compared to wounds treated with the other ointments. This study suggested that using 0.2 wt% BiSCaO ointment as a disinfectant for infected wounds and limiting disinfection to 3 days may be sufficient to avoid the negative effects of BiSCaO on wound repair.

Safety of Concentrated Bioshell Calcium Oxide Water Application for Surface and Skin Disinfections against Pathogenic Microbes.

Immediately post-production, commercially available bioshell calcium oxide (BiSCaO) water is colorless, transparent, and strongly alkaline (pH 12.8), and is known to possess deodorizing properties and broad microbicidal activity. However, BiSCaO Water may represent a serious safety risk to the living body, given the strong alkalinity. This study aimed to investigate the safety of BiSCaO Water for use as an antiseptic/disinfectant despite concerns regarding its high alkalinity. The change over time in pH of BiSCaO Water was measured during air contact (stirring BiSCaO Water in ambient air). When sprayed on metal, plastic, wood piece, paper, and skin surfaces, the pH of BiSCaO Water decreased rapidly, providing a white powder coating upon drying. Scanning electron microscopy images, energy dispersive X-ray elemental mapping, and X-ray diffractograms showed that the dried powder residues of BiSCaO Water were composed primarily of calcium carbonate. These results suggested that BiSCaO Water is a potent reagent that may overcome the obstacles of being strongly alkaline, making this material appropriate for use in disinfection against pathogenic microbes.

Heparinoid Complex-Based Heparin-Binding Cytokines and Cell Delivery Carriers.

Heparinoid is the generic term that is used for heparin, heparan sulfate (HS), and heparin-like molecules of animal or plant origin and synthetic derivatives of sulfated polysaccharides. Various biological activities of heparin/HS are attributed to their specific interaction and regulation with various heparin-binding cytokines, antithrombin (AT), and extracellular matrix (ECM) biomolecules. Specific domains with distinct saccharide sequences in heparin/HS mediate these interactions are mediated and require different highly sulfated saccharide sequences with different combinations of sulfated groups. Multivalent and cluster effects of the specific sulfated sequences in heparinoids are also important factors that control their interactions and biological activities. This review provides an overview of heparinoid-based biomaterials that offer novel means of engineering of various heparin-binding cytokine-delivery systems for biomedical applications and it focuses on our original studies on non-anticoagulant heparin-carrying polystyrene (NAC-HCPS) and polyelectrolyte complex-nano/microparticles (N/MPs), in addition to heparin-coating devices.

Application of hydrogels as submucosal fluid cushions for endoscopic mucosal resection and submucosal dissection.

Numerous new techniques have recently been reported and described for the endoscopic mucosal resection (EMR) of large superficial lesions of the gastrointestinal tract, using various natural, synthetic, and semi-synthetic materials such as chitin/chitosan and their derivatives. Although saline-assisted EMR is an established minimally invasive therapy, en bloc resection and histopathological analyses are required to determine its curative potential. In addition, complete resection of lesions of >2 cm in diameter remains difficult, despite improved EMR techniques. The development of endoscopic submucosal dissection (ESD) has increased dissection rates for en bloc resection of large lesions, but perforation occurs more frequently during ESD than during EMR. Submucosal injections of those biomaterials which have high viscosity and hydrogelatinization ability as submucosal fluid cushions (SFC) may facilitate ESD as well as EMR for the treatment of superficial tumors of the alimentary tract. In this review, we describe the application of biomaterials such as chitosan derivatives, sodium hyaluronate, and 50% glucose as a SFC for ESD, focusing photocrosslinked chitosan hydrogels (PCH) which we have originally developed.

Biomedical application of low molecular weight heparin/protamine nano/micro particles as cell- and growth factor-carriers and coating matrix.

Low molecular weight heparin (LMWH)/protamine (P) nano/micro particles (N/MPs) (LMWH/P N/MPs) were applied as carriers for heparin-binding growth factors (GFs) and for adhesive cells including adipose-derived stromal cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs). A mixture of LMWH and P yields a dispersion of N/MPs (100 nm-3 µm in diameter). LMWH/P N/MPs can be immobilized onto cell surfaces or extracellular matrix, control the release, activate GFs and protect various GFs. Furthermore, LMWH/P N/MPs can also bind to adhesive cell surfaces, inducing cells and LMWH/P N/MPs-aggregate formation. Those aggregates substantially promoted cellular viability, and induced vascularization and fibrous tissue formation in vivo. The LMWH/P N/MPs, in combination with ADSCs or BMSCs, are effective cell-carriers and are potential promising novel therapeutic agents for inducing vascularization and fibrous tissue formation in ischemic disease by transplantation of the ADSCs and LMWH/P N/MPs-aggregates. LMWH/P N/MPs can also bind to tissue culture plates and adsorb exogenous GFs or GFs from those cells. The LMWH/P N/MPs-coated matrix in the presence of GFs may provide novel biomaterials that can control cellular activity such as growth and differentiation. Furthermore, three-dimensional (3D) cultures of cells including ADSCs and BMSCs using plasma-medium gel with LMWH/P N/MPs exhibited efficient cell proliferation. Thus, LMWH/P N/MPs are an adequate carrier both for GFs and for stromal cells such as ADSCs and BMSCs, and are a functional coating matrix for their cultures.

Development of antimicrobial biomaterials produced from chitin-nanofiber sheet/silver nanoparticle composites.

BACKGROUND: Chitin nanofibers sheets (CNFSs) with nanoscale fiber-like surface structures are nontoxic and biodegradable biomaterials with large surface-to-mass ratio. CNFSs are widely applied as biomedical materials such as a functional wound dressing. This study aimed to develop antimicrobial biomaterials made up of CNFS-immobilized silver nanoparticles (CNFS/Ag NPs). MATERIALS AND METHODS: CNFSs were immersed in suspensions of Ag NPs (5.17 ± 1.9 nm in diameter; mean ± SD) for 30 min at room temperature to produce CNFS/Ag NPs. CNFS/Ag NPs were characterized by transmission electron microscopy (TEM) and then tested for antimicrobial activities against Escherichia (E.) coli, Pseudomonas (P.) aeruginosa, and H1N1 influenza A virus, three pathogens that represent the most widespread infectious bacteria and viruses. Ultrathin sectioning of bacterial cells also was carried out to observe the bactericidal mechanism of Ag NPs. RESULTS: The TEM images indicated that the Ag NPs are dispersed and tightly adsorbed onto CNFSs. Although CNFSs alone have only weak antimicrobial activity, CNFS/Ag NPs showed much stronger antimicrobial properties against E. coli, P. aeruginosa, and influenza A virus, with the amount of immobilized Ag NPs onto CNFSs. CONCLUSIONS: Our results suggest that CNFS/Ag NPs interacting with those microbes exhibit stronger antimicrobial activities, and that it is possible to apply CNFS/Ag NPs as anti-virus sheets as well as anti-infectious wound dressings.

Nanospiked paper: Microfibrous cellulose materials nanostructured via partial hydrolysis and self-assembly.

Nanocelluloses, such as cellulose nanofibers and nanocrystals, are sustainable nanomaterials that are generally extracted from natural raw materials in a top-down manner. These nanomaterials and their assemblies are facilitating new applications of biopolymers. However, creating nanostructures from conventional cellulosic materials including paper and cloth remains challenging. Herein, we report an approach for bottom-up nanostructuring of conventional microfibrous cellulose materials via a molecular self-assembly strategy. As a precursor cellulose material, paper was allowed to swell with aqueous phosphoric acid for the partial dissolution and hydrolysis of cellulose while maintaining its microfibrous structure. The generated cello-oligosaccharides in a dissolved state started to self-assemble upon adding water as a coagulant, resulting in nanospike-like assemblies on the microfiber surfaces. The resultant nanospiked papers were found to serve as a precursor for synthesizing silver nanoparticle-cellulose composites with bactericidal activities. Our findings promote the development of cellulose-based functional materials with nanostructures designed via molecular self-assembly.

Endoscopic submucosal dissection for pig esophagus by using photocrosslinkable chitosan hydrogel as submucosal fluid cushion.

BACKGROUND: Hypertonic saline solution (HS) as a submucosal fluid cushion (SFC) for endoscopic submucosal dissection (ESD) has several disadvantages, including a short effect duration and increased risk of bleeding and perforation. Photocrosslinkable chitosan hydrogel in DMEM/F12 medium (PCH) can be converted into an insoluble hydrogel by UV irradiation for 30 seconds. OBJECTIVE: To evaluate the feasibility, usefulness, and safety of PCH as an SFC for ESD of esophagi, compared with HS and sodium hyaluronate (SH). DESIGN: Survival animal study. SETTINGS: Research laboratory study of 24 pig models in vivo. INTERVENTIONS: Twenty-four pigs were used in the 2 steps: First, ESD of the esophagus was performed with PCH, SH, or HS (each n = 6) as an SFC, and the effects of these agents on wound healing were examined endoscopically and histologically. Second, in vivo degradation of PCH (n = 3) and HS (n = 3) was examined in independent pig esophagi. MAIN OUTCOME MEASUREMENTS: Outcome measurements included feasibility and safety of PCH-assisted ESD of esophagus, gross and histologic evidence of the treated esophagus, biodegradation of injected PCH, and clinical tolerance by the animals. RESULT: PCH injection led to a longer-lasting elevation with clearer margins compared with SH and HS, thus enabling precise ESD along the margins of the elevated mucosa without complications such as bleeding and perforation. The aspects of wound repair after PCH-assisted ESD were similar to those of SH- and HS-assisted ESDs. Biodegradation of PCH was confirmed to be almost completed within 8 weeks on the basis of endoscopic and histologic observations. LIMITATIONS: In vivo animal model study. CONCLUSION: PCH permits more reliable ESD of the esophagus without complications than do SH and HS. Furthermore, the applied PCH appeared to be completely biodegraded within 8 weeks. Thus, PCH is a promising agent as an SFC in ESD of the esophagus.

Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with hydrosheets composed of chitin/chitosan, fucoidan, and alginate as wound dressings.

To create a moist environment for rapid wound healing, a hydrosheet composed of alginate, chitin/chitosan, and fucoidan (ACF-HS) has been developed as a functional wound dressing. The aim of this study was to evaluate the accelerating effect of ACF-HS on wound healing for rat mitomycin C-treated healing-impaired wounds. Full-thickness skin defects were made on the back of rats and mitomycin C was applied onto the wound for 10 minutes to prepare a healing-impaired wound. After thoroughly washing out the mitomycin C, ACF-HS was applied to the healing-impaired wounds. The rats were later euthanized and histological sections of the wounds were prepared. The histological examinations showed significantly advanced granulation tissue and capillary formations in the healing-impaired wounds treated with ACF-HS on days 7 and 14, in comparison with that in alginate fiber (Kaltostat), hydrogel wound dressing (DuoACTIVE), and nontreatment (negative control). Furthermore, in cell culture studies, ACF-HS-absorbed serum and fibroblast growth factor-2 was found to be proliferative for fibroblasts and endothelial cells, respectively, and ACF-HS-absorbed serum was found to be chemoattractive for fibroblasts. However, our results may not be strictly comparable with general healing-impaired wound models in humans because of the cell damage by mitomycin C. In addition, more biocompatibility studies of fucoidan are essential due to the possibility of renal toxicity.

Bioshell calcium oxide (BiSCaO) for cleansing and healing Pseudomonas aeruginosa-infected wounds in hairless rats.

BACKGROUND: Scallop shell powder is called bioshell calcium oxide (BiSCaO), which is known to possess deodorizing properties and broad antimicrobial activity against various pathogenic microbes, including viruses, bacteria, spores, and fungi. OBJECTIVE: This study aims to investigate the applications of BiSCaO suspension cleansing in clinical situations, for instance for the prevention and treatment of infections in chronic wounds in healing-impaired patients, without delaying wound healing. METHODS: The bactericidal activities of 1000 ppm BiSCaO suspension; 500 ppm hypochlorous acid; 1000 ppm povidone iodine; and saline were compared to evaluate in vivo disinfection and healing of Pseudomonas aeruginosa-infected wounds in hairless rats. RESULTS: Cleansing of the infected wounds with BiSCaO suspension daily for 3 days significantly enhanced wound healing and reduced the in vivo bacterial counts, in comparison to hypochlorous acid, povidone iodine, and saline. Furthermore, histological examinations showed significantly advanced granulation tissue and capillary formation in the wounds cleansed with BiSCaO suspension than in those cleansed with the other solutions. CONCLUSIONS: This study suggested that the possibility of using BiSCaO suspension as a disinfectant for infected wounds and limiting disinfection to 3 days may be sufficient to avoid the negative effects on wound repair.

Coatings of low-density lipoprotein and synthetic glycoconjugates as substrata for hepatocytes.

Asialoglycoprotein (ASGP) receptors expressed on rat hepatocytes interact with glycoproteins containing galactose or N-acetylgalactosamine residues at the nonreducing termini of oligosaccharide chains to mediate endocytosis, and cholesterol transport protein with apolipoprotein B (LDL, low-density lipoprotein) in plasma interacts with LDL receptors and heparinoids in the extracellular matrix. We developed novel techniques to prepare galactose- and LDL-immobilized culture plates, using galactose-tagged polystyrene (galactose-carrying polystyrene [GalCPS]: N-p-vinylbenzyl-O-beta-D-galactopyranosyl-[1-->4]-D-gluconamide) and poly(2-acrylamide-2-methyl-1-propanesulfonate) (PAPS), respectively. Hepatocytes adhered well to plates coated with either GalCPS or LDL, and therefore the GalCPS- and LDL-coated plates were examined as specific substrata for culturing hepatocytes. These cultures promoted the formation of three-dimensional, multicellular aggregates with regulation of excess proliferation of non-parenchymal cells. Furthermore, the LDL coating resulted in higher albumin synthesis and an identical level of lactate dehydrogenase (LDH) compared with cells cultured on collagen- and GalCPS-coated plates. Thus, the two culture systems described here, and especially the LDL-coated plates, have potential for the development of a hybrid artificial liver.

Accelerated wound healing in healing-impaired db/db mice by autologous adipose tissue-derived stromal cells combined with atelocollagen matrix.

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.

Biomaterials as cell carriers for augmentation of adipose tissue-derived stromal cell transplantation.

Adipose tissue-derived stromal cells (ADSCs) contain lineage-committed progenitor cells that have the ability to differentiate into various cell types that may be useful for autologous cell transplantation to correct defects of skin, adipose, cartilage, bone, tendon, and blood vessels. The multipotent characteristics of ADSCs, as well as their abundance in the human body, make them an attractive potential resource for wound repair and applications to tissue engineering. ADSC transplantation has been used in combination with biomaterials, including cell sheets, hydrogel, and three-dimensional (3D) scaffolds based on chitosan, fibrin, atelocollagen, and decellularized porcine dermis, etc. Furthermore, low molecular weight heparin/protamine nanoparticles (LH/P NPs) have been used as an inducer of ADSC aggregation. The tissue engineering potential of these biomaterials as cell carriers is increased by the synergistic relationship between ADSCs and the biomaterials, resulting in the release of angiogenic cytokines and growth factors. In this review article, we describe the advantages of ADSC transplantation for tissue engineering, focusing on biomaterials as cell carriers which we have studied.

Transplantation of inbred adipose-derived stromal cells in rats with plasma gel containing fragmin/protamine microparticles and FGF-2.

Fragmin/protamine microparticles (F/P MPs) have been used as a cell carrier for adipose-derived stromal cells (IR-ASCs) in inbred male Fisher 344 rats, and for preservation and controlled-release of fibroblast growth factor (FGF)-2 and various cytokines in inbred rat plasma (IRP)-DMEM (Dulbecco's modified Eagle's medium) gel. In this study, we investigated the capability of an IRP-DMEM gel containing F/P MPs and/or FGF-2, as a three-dimensional (3D)-culture, to expand IR-ASCs. We found that IR-ASCs grow faster under 3D-culture conditions in low IRP (3%)-DMEM gel containing F/P MPs and FGF-2 without any animal serum than those under 2D-culture in low inbred rat serum (3%)-DMEM with F/P MPs and FGF-2. About 0.3 mL of IR-ASCs (about 4,000,000 cells mL⁻¹) grown in IRP (6%)-DMEM gel containing F/P MPs and FGF-2 disappeared 8 days after subcutaneous injection in rats, suggesting that they are rapidly biodegradable. The number of large (diameter ≥200 µm or containing ≥100 erythrocytes), medium (diameter = 20-200 µm or containing 10-100 erythrocytes) and small (diameter ≤20 µm or containing 1-10 erythrocytes) capillaries after injection with IR-ASCs in an IRP-DMEM gel containing both F/P MPs and FGF-2, as well as the thickness of tissue granulation per microphotograph at the injected site, was significantly higher than those after injection with IR-ASCs in an IRP-DMEM gel containing either FGF-2 or F/P MPs. Thus, IRP-DMEM gel containing F/P MPs and FGF-2 are useful and safe IR-ASC carriers that facilitate cell proliferation, vascularization, and tissue granulation locally at injection sites.

Midterm results of the "sandwich technique" via a right ventricle incision to repair post-infarction ventricular septal defect.

BACKGROUND: Residual shunting and mortality are problems associated with current surgical repair techniques for post-infarction ventricular septal defects. METHODS: We describe the mid-term results of the "sandwich technique" to repair a post-infarction ventricular septal defect (VSD), performed via a right ventricle incision. Application of direct ultrasonography to the right ventricular wall enables a surgeon to visualize the region, perform an appropriate incision into the right ventricle, and perform a trabecula resection. One patch is placed on the left ventricular (LV) side and the other on the right ventricular (RV) side of the VSD. The VSD is sealed with gelatin-resorcin-formalin (GRF) glue between the two patches. RESULTS: We had seven consecutive patients. The sandwich technique resulted in geometric preservation of the LV shape. There were no significant leaks, no mortality within a thirty-day postoperative period, and no bleeding problems. Hospital mortality was 14.3% (1/7 cases). Late survival longer than a year was obtained in five cases (71%). The longest patient survival time was nine years. No tissue degeneration was noted. CONCLUSION: This technique may be useful for repairing a post-infarction VSD.

The "loop with anchor" technique to repair mitral valve prolapse.

The current surgical technique of using an artificial chord (composed of expanded polytetrafluoroethylene [ePTFE] sutures) to repair mitral prolapse is technically difficult to perform. Slippery knot tying and the difficulty of changing the chordae length after the hydrostatic test are frustrating problems. The loop technique solves the problem of slippery knot tying but not the problem of changing the chordae length. Our "loop with anchor" technique consists of the following elements: construction of an anchor at the papillary muscle; determining the loop length; tying the loop to the anchor; suturing the loop to the mitral valve; the hydrostatic test; and re-suturing or changing the loop, if needed. Adjustments can be made for the entire procedure or for a portion of the procedure.

In vitro study of the optimum volume ratio of activator to adhesive in gelatin-resorcin-formalin glue.

OBJECTIVE: Excessive use of activator (formaldehyde + glutaraldehyde) may cause late complications after use of gelatin-resorcin-formalin (GRF) glue during surgery. The goal of the study was to define the appropriate ratio of activator to adhesive and to establish an approach for accurate control of this ratio. METHODS: The relation between adhesive force and the activator/adhesive ratio was studied by attaching two sheets of equine pericardium using GRF glue, with ratios from 1: 50 to 1: 2. The amount of activator was measured per drip from the needle in the GRF glue kit and other needles (27, 25, 23, 22, and 21 gauge). RESULTS: The adhesive force was about 400 gram-weight (gw) for activator/adhesive ratios from 1: 50 to 1: 20. This force showed a significant increase to 1317 ± 462 gw for a ratio of 1: 10 compared to the force at a ratio 1: 20 (P = 0.0069) but did not increase significantly for ratios above 1: 10. The activator volume was 12.5 µl in one drip from the needle in the GRF glue kit and 4.3 µl in one drip from the 27-gauge needle. Therefore, the 27-gauge needle is suitable for measuring the activator volume. CONCLUSION: In vitro, an activator at a ratio of one-tenth the volume of the adhesive provides approximately maximum force; any more activator is residual and potentially harmful. Measurement of the activator volume using a 27-gauge needle and the adhesive volume using a syringe is recommended to control the ratio accurately.

Fragmin/protamine microparticles as cell carriers to enhance viability of adipose-derived stromal cells and their subsequent effect on in vivo neovascularization.

We prepared fragmin/protamine microparticles (F/P MPs) as cell carriers to enhance cell viability. Use of material consisting of a low-molecular-weight heparin (fragmin) mixed with protamine resulted in water-insoluble microparticles (about 0.5-1 microm in diameter). In this study, we investigated the capability of F/P MPs to enhance the viabilities of human microvascular endothelial cells (HMVECs), human dermal fibroblasts (fibroblasts), and adipose tissue-derived stromal cells (ATSCs) in suspension culture. F/P MPs were bound to the surfaces of these cells, and the interaction of these cells with F/P MPs induced cells/F/P MPs-aggregate formations in vitro, and maintained viabilities of those cells for at least 3 days. The ATSCs/F/P MPs-aggregates adhered to and grew on suspension culture plates in a fashion similar to those on type I collagen-coated plates. The cultured ATSCs secreted significant amounts of angiogenic heparin-binding growth factors such as FGF-2. When the ATSCs/F/P MPs-aggregates were subcutaneously injected into the back of nude mice, significant neovascularization and fibrous tissue formation were induced near the site of injection from day 3 to week 2. The ATSCs/F/P MPs-aggregates were thus useful and convenient biomaterials for cell-therapy of angiogenesis.

Expansion and characterization of human bone marrow-derived mesenchymal stem cells cultured on fragmin/protamine microparticle-coated matrix with fibroblast growth factor-2 in low serum medium.

Fragmin/protamine microparticles (F/P MPs) can be stably coated onto plastic surfaces. A capability of F/P MP-coated plates was investigated to immobilize fibroblast growth factor (FGF)-2 as a substratum to expand human bone marrow-derived mesenchymal stem cells (BMMSCs). FGF-2 molecules in low (2%) human serum (HS) medium were immobilized onto F/P MP-coated plates, and the FGF-2 was gradually released into the medium with a half-releasing time of 4-5 days. BMMSCs adhered well to the F/P MP-coated plates, and grew at a doubling time of about 28 h in low (2%) HS medium with FGF-2 (5 ng/mL), while the cells grew at a doubling time of about 30 and 38 h in high (10%) HS medium and in low (2%) HS medium with FGF-2, respectively, without F/P MP coating. The expanded BMMSCs on the F/P MP-coated plates in low (2%) HS medium with FGF-2 maintained their multilineage potential for differentiation into adipocytes and osteoblasts.

Effect of controlled release of fibroblast growth factor-2 from chitosan/fucoidan micro complex-hydrogel on in vitro and in vivo vascularization.

We produced a chitosan/fucoidan micro complex-hydrogel as a carrier for controlled release of heparin binding growth factors such as fibroblast growth factor (FGF)-2. Material consisting of a soluble chitosan (CH-LA) mixed with fucoidan yielded a water-insoluble and injectable hydrogel with filamentous particles. In this study, we examined the ability of the chitosan/fucoidan complex-hydrogel to immobilize FGF-2 and to protect its activity, as well as the controlled release of FGF-2 molecules. The chitosan/fucoidan complex-hydrogel has high affinity for FGF-2 (K(d) = 5.4 x 10(-) (9)M). The interaction of FGF-2 with chitosan/fucoidan complex-hydrogel substantially prolonged the biological half-life time of FGF-2. It also protected FGF-2 from inactivation, for example by heat and proteolysis, and enhance FGF-2 activity. When FGF-2-containing complex-hydrogel was subcutaneously injected into the back of mice, significant neovascularization and fibrous tissue formation were induced near the site of injection at 1 week, and the complex-hydrogel was biodegraded and disappeared by 4 weeks. These findings indicate that controlled release of biologically active FGF-2 molecules is caused by both slow diffusion and biodegradation of the complex-hydrogel, and that subsequent induction of vascularization occurs. FGF-2-containing chitosan/fucoidan micro complex-hydrogel is thus useful and convenient for treatment of ischemic disease.