Satoko Kishimoto

Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-impaired wound dressings.

In order to create a moist environment for rapid wound healing, a hydrogel sheet composed of a blended powder of alginate, chitin/chitosan and fucoidan (ACF-HS; 60:20:2:4 w/w) has been developed as a functional wound dressing. ACF-HS gradually absorbed DMEM without any maceration, and fluid absorption became constant within 18 h. On application, ACF-HS was expected to effectively interact with and protect the wound in rats, providing a good moist healing environment with exudates. In addition, the wound dressing has properties such as ease of application and removal and good adherence. Full-thickness skin defects were made on the backs of rats and mitomycin C solution (1 mg/ml in saline) was applied onto the wound for 10 min in order to prepare healing-impaired wounds. After thoroughly washing out the mitomycin C, ACF-HS was applied to the healing-impaired wounds. Although normal rat wound repair was not stimulated by the application of ACF-HS, healing-impaired wound repair was significantly stimulated. Histological examination demonstrated significantly advanced granulation tissue and capillary formation in the healing-impaired wounds treated with ACF-HS on day 7, as compared to those treated with calcium alginate fiber (Kaltostat; Convatec Ltd., Tokyo, Japan) and those left untreated.

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.

Enhanced Effect of Platelet-Rich Plasma Containing a New Carrier on Hair Growth

BACKGROUND Treatments for alopecia are in high demand, but not all are safe and reliable. Dalteparin and protamine microparticles (D/P MPs) can effectively carry growth factors (GFs) in platelet‐rich plasma (PRP). OBJECTIVE To identify the effects of PRP‐containing D/P MPs (PRP&D/P MPs) on hair growth. METHODS & MATERIALS Participants were 26 volunteers with thin hair who received five local treatments of 3 mL of PRP&D/P MPs (13 participants) or PRP and saline (control, 13 participants) at 2‐ to 3‐week intervals and were evaluated for 12 weeks. Injected areas comprised frontal or parietal sites with lanugo‐like hair. Experimental and control areas were photographed. Consenting participants underwent biopsies for histologic examination. RESULTS D/P MPs bind to various GFs contained in PRP. Significant differences were seen in hair cross‐section but not in hair numbers in PRP and PRP&D/P MP injections. The addition of D/P MPs to PRP resulted in significant stimulation in hair cross‐section. Microscopic findings showed thickened epithelium, proliferation of collagen fibers and fibroblasts, and increased vessels around follicles. CONCLUSION PRP&D/P MPs and PRP facilitated hair growth but D/P MPs provided additional hair growth. The authors have indicated no significant interest with commercial supporters.

Enhanced healing of mitomycin C-treated wounds in rats using inbred adipose tissue-derived stromal cells within an atelocollagen matrix.

The aim of this study was to evaluate the potential accelerating effects of an adipose tissue‐derived stromal cells (ATSC)‐containing atelocollagen matrix with silicone membrane (ACMS) for repairing mitomycin C‐treated healing‐impaired wounds. Mitomycin C was applied to full‐thickness skin incisions in this study to create a healing‐impaired wound model in rat. After thoroughly washing out the mitomycin C from the wound, ACMS alone or ATSC‐containing ACMS was applied to the wounds. Histological sections of the wounds were then prepared at indicated time periods after the treatments. These results indicated significantly advanced granulation tissue and capillary formations in the healing‐impaired wounds treated with ATSC‐containing ACMS compared with those treated with ACMS alone. Thus, this study suggested that transplantation of inbred ATSC‐containing ACMS is effective for repairing healing‐impaired wounds.

Platelet-rich Plasma (prp) Promotes Survival of Fat-grafts in Rats

This study evaluated the effects of platelet-rich plasma (PRP) on resorption and adipocyte survival in autologous fat-graft of rats prepared with isogenous PRP. Fat grafts prepared without PRP (control group) became united to the tissue adjacent to the implantation site and were significantly resorbed from 30 days. On the other hand, fat grafts prepared with PRP (PRP group) demonstrated little resorption from 30 to 120 days and appeared pink, had a soft, supple feel, and were easily compressible. Histologic sections of grafts in the control and PRP groups at 10 days exhibited similar consolidation of the grafted tissue, which contained morphologically normal adipocytes with different degrees of granulation and capillary formation. From 20 days normal adipocytes were obviously decreased in the control group, while the PRP group demonstrated increased granulation tissue and capillary formation and good maintenance of normal adipocytes for at least 120 days.

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

Controlled release of FGF-2 using fragmin/protamine microparticles and effect on neovascularization

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.

Preparation and characterization of low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as FGF-2 carrier

We produced low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as a carrier for heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2). A mixture of low-molecular-weight heparin (MW: about 5000 Da, 6.4 mg/mL) and protamine (MW: about 3000 Da, 10 mg/mL) at a ratio of 7:3 (vol:vol) yields a dispersion of microparticles (1–6 μm in diameter). In this study, diluted low-molecular-weight heparin solution in saline (0.32 mg/mL) mixed with diluted protamine (0.5 mg/mL) at a ratio at 7:3 (vol:vol) resulted in soluble nanoparticles (112.5 ± 46.1 nm in diameter). The generated NPs could be then stabilized by adding 2 mg/mL dextran (MW: 178–217 kDa) and remained soluble after lyophilization of dialyzed LMW-H/P NP solution. We then evaluated the capacity of LMW-H/P NPs to protect activity of FGF-2. Interaction between FGF-2 and LMW-H/P NPs substantially prolonged the biological half-life of FGF-2. Furthermore, FGF-2 molecules were protected from inactivation by heat and proteolysis in the presence of LMW-H/P NPs.

Preparation of Size-Controlled Silver Nanoparticles and Chitin-Based Composites and Their Antimicrobial Activities

A simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) was reported for their generation by autoclaving a mixture of silver-containing glass powder and glucose. The particle size is regulated by the glucose concentration, with concentrations of 0.25, 1.0, and 4.0 wt% glucose providing small ( nm in diameter), medium ( nm), and large ( nm) particles, respectively. In this study, Ag NP/chitin composites were synthesized by mixing each of these three Ag NP suspensions with a <5% deacetylated (DAc) chitin powder (pH 7.0) at room temperature. The Ag NPs were homogenously dispersed and stably adsorbed onto the chitin. The Ag NP/chitin composites were obtained as yellow or brown powders. Approximately 5, 15, and 20 μg of the small, medium, and large Ag NPs, respectively, were estimated to maximally adsorb onto 1 mg of chitin. The bactericidal and antifungal activities of the Ag NP/chitin composites increased as the amount of Ag NPs in the chitin increased. Furthermore, smaller Ag NPs (per weight) in the chitin composites provided higher bactericidal and anti-fungal activities.

Enhancement of vascularization and granulation tissue formation by growth factors in human platelet‐rich plasma‐containing fragmin/protamine microparticles

The purpose of this study was to evaluate effects of human platelet-rich plasma (PRP)-containing fragmin/protamine microparticles (F/P MPs) as a protein carrier on neovascularization and granulation tissue formation. Frozen and thawed PRP contains high concentrations of various growth factors (GFs) and F/P MPs effectively adsorb those GFs. Human microvascular endothelial cells (MVECs) and dermal fibroblast cells (DFCs) were optimally grown in medium containing 4% PRP and the addition of F/P MPs significantly maintained and protected the proliferative activity of PRP incubated at 37°C for more than 10 days. When PRP-containing F/P MPs were subcutaneously injected into the back of mice, significant neovascularization was induced near the injected site with enhanced filtration of inflammatory cells from day 3 to day 30, compared with controls (injections of PRP, F/P MPs, and saline). Both PRP-containing F/P MPs and PRP alone induced significant formation of granulation tissue at the injected site. However, thickness of induced granulation tissues was well maintained for 30 days only in PRP-containing F/P MP-injected group. Those bound GFs may be gradually diffused and released from F/P MPs in vitro and in vivo. Thereby, PRP-containing F/P MPs offer significantly higher inductions of vascularization and fibrous tissue formation in vivo than PRP alone.