Megumi Takikawa

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.

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.

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.

Detection of skin perforators by indocyanine green fluorescence nearly infrared angiography.

Perforator-based island flaps are widely used to reconstruct skin defects. For this procedure to succeed, a perforator with adequate blood flow must be selected, and precise preoperative prediction of the location of the perforators is required. To identify perforators, a variety of methods have bee

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.

PRP&F/P MPs Improved Survival of Dorsal Paired Pedicle Skin Flaps in Rats

BACKGROUND Skin flap necrosis is a problem encountered postoperatively. The purpose of this study was to evaluate the effects of platelet-rich plasma containing fragmin/protamine microparticles (PRP&F/P MPs) on viability in a rat dorsal paired pedicle skin (DPPS) flap. MATERIALS AND METHODS Two symmetrical adjoining rectangular flaps (8 × 2 cm each) were drawn on the rat dorsum. Two days after PRP&F/P MPs-, PRP-, F/P MPs-, and saline (control)-injections (n = 8 each), flaps were elevated as a random pattern flap without the lateral thoracic, posterior intercostal, and deep circumflex iliac vessels. The flaps were immediately sutured back and the flap survival area was measured 7 d after flap elevation. RESULTS The flap survival rate in PRP&F/P MPs-injected groups (73.1% ± 4.2%) was significantly higher than those in PRP (64.9% ± 4.0%), F/P MPs (59.4 ± 4.5%), and control (61.2% ± 4.2%) groups. Histologic observation of the flaps showed survived thick granulation tissue and neovascularization in PRP&F/P MPs-injected groups. CONCLUSIONS When PRP&F/P MPs are administered 2 d before the flap elevation, the improved flap survivals are observed. The pre-injection of PRP&F/P MPs may thus represent a promising treatment to prevent skin flap necrosis in reconstructive surgery.

Effective expansion of human adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles-coated substratum with human platelet-rich plasma.

Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet‐rich plasma (PRP). In this study, we investigated the capability of F/P NP‐coated plates to act as a substratum for the proliferation of human adipose‐derived stromal cells (ASCs) and bone marrow‐derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP‐coated plates and grew optimally, with a doubling time of 30 and 32 h in low‐concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor‐2 (FGF‐2) on the F/P NP‐coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF‐2 on F/P NP‐coated plates pretreated with PRP and FGF‐2 in a concentration‐dependent manner. Thus, F/P NP‐coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low‐concentration PRP medium supplemented with FGF‐2. No xenogeneic serum is required. Copyright

Increased survival of free fat grafts and vascularization in rats with local delivery of fragmin/protamine microparticles containing FGF-2 (F/P MP-F)

We evaluated the effects of fragmin/protamine micro-particles (F/P MPs) containing FGF-2 (F/P MP-F) as carriers for the controlled release of FGF-2 for adipocyte-survival and capillary formation in inbred rats with subdivided free fat grafts. F/P MPs could immobilize FGF-2, thereafter gradually releasing the bound FGF-2. Inbred Fisher 344 rats weighing around 150 g were anesthetized and implanted with paste comprising harvested fat combined with F/P MP-F. The effect of F/P MP-F on the survival, granulation, and capillary formation in fat grafts was histologically compared with control grafts containing either FGF-2, F/P MPs or PBS. The control fat grafts became attached to tissues adjacent to the implantation site and were significantly resorbed after 30 days. In contrast, pink, soft, supple grafts were compressible and were little resorbed in the group given F/P FP MP-F at 30-120 days. Normal adipocytes were obviously decreased in the control groups with increased granulation tissues, whereas normal adipocytes with capillary formations were maintained in the F/P MP-F group. Thus, adding F/P MP-F to subdivided fat grafts helps to improve graft volume retention and survival in soft-tissue reconstruction through accelerating adipocyte-survival rates and angiogenesis.

Fragmin/protamine microparticles to adsorb and protect HGF and to function as local HGF carriers in vivo

The clinical efficacy of hepatocyte growth factor (HGF) in tissue repair can be greatly enhanced by high affinity, biocompatible drug carriers that maintain the bioactivity and regulate release at the target site. We produced 0.5-3.0 μm fragmin (low molecular weight heparin)/protamine microparticles (F/P MPs) as carriers for the controlled release of HGF. F/P MPs immobilized more than 3 μg of HGF per mg of MPs and gradually released the absorbed HGF into the medium with a half-release time of approximately 5 days. Compared with HGF alone, HGF-containing F/P MPs substantially enhanced the mitogenic effect of HGF on cultured human microvascular endothelial cells, by prolonging the biological half-life, and its conjugation to F/P MPs protected HGF from heat and proteolytic inactivation. F/P MPs disappeared 8 days after subcutaneous injection in mice, suggesting that they are rapidly biodegraded. Furthermore, the number of large (diameter ≥200 μm or containing ≥ 100 erythrocytes) and medium (diameter 20-200 μm or containing 10-100 erythrocytes) lumen capillaries 8 days after injection of HGF-containing F/P MPs was significantly higher than that after injection of HGF or F/P MPs alone. Furthermore, the number of small (diameter ≤ 20 μm or containing 1-10 erythrocytes) lumen capillaries was significantly higher 4 days after injection of HGF-containing F/P MPs. This increased angiogenic activity of HGF in vivo is probably due to both sustained local release and protection against biodegradation by the F/P MPs. Thus, F/P MPs may be useful and safe HGF carriers that facilitate cell proliferation and vascularization at sites of tissue damage.

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.