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Background Chitosan has been investigated as a promising nonviral vector. However, several problems still remain, such as a relatively low transfection efficiency and instability under physiological conditions. We previously demonstrated that a chondroitin sulfate (CS) coating enhanced the transfection efficiency and physicochemical stability of plasmid DNA (pDNA)/chitosan complexes in vitro. In the present study, the effects of coating pDNA/chitosan complexes with CS on the stability in freeze–dry rehydration processes and gene expression in vivo were investigated. Methods Freeze-drying storage at –20 °C, 4 °C, or room temperature, freezing storage at –20 °C, or liquid storage at 4 °C or room temperature, were examined for preservation conditions of pDNA/chitosan/CS ternary complexes by a gel retardation assay, measurements of sizes and zeta potentials, and a luciferase assay. Moreover, to determine the transfection efficiency of the ternary complexes in vivo, suicide gene therapy was carried out in Huh-7-implanted mice using herpes simplex virus thymidine kinase coding pDNA and ganciclovir. Results The freeze-dried pDNA/chitosan/CS ternary complexes showed sufficient cell transfection ability in vitro and in vivo. In addition, ternary complexes were associated with a significant suppression of tumor growth and a histopathologically high anti-tumor effect by intratumoral injection to tumor-bearing mice. Conclusions The CS coating enhanced the preservation stability of the pDNA/chitosan complexes after freeze-drying–rehydration and their transgene expression in vivo. Copyright © 2013 John Wiley & Sons, Ltd.

Systematic structural studies of 24 different kinds of endohedral metallofullerenes, M(x)C(2n) (M = La, Y, Sc, Lu, Ti, Eu, Er, Hf, Sc(3)N; 34 ≤ n ≤ 43), as 1:1 cocrystals with solvent toluene molecules have been carried out using synchrotron radiation powder diffraction. Thirteen of the 24 molecular structures, including five metal carbides, one metal nitride endohedral fullerene, and one hollow fullerene, have been determined by a combination of the maximum entropy method and Rietveld refinement of the X-ray diffraction data obtained. We have found that the volume for one fullerene and one toluene molecule depends linearly on the number of carbon atoms in the fullerene cage. Fifteen different kinds of metal carbide endohedral fullerenes have been identified, which can be structurally characterized from the obtained lattice constants using only this linear dependence. The linear dependence found in the present study provides a metallofullerene diagnostics diagram that may have universal importance for structural characterization of the so-called cluster endohedral fullerenes.

We previously reported a simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) generated 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 (3.48 ± 1.83 nm in diameter), medium (6.53 ± 1.78 nm) and large (12.9 ± 2.5 nm) particles, respectively. In this study, Ag NP/chitosan composites were synthesized by mixing each of these three Ag NP suspensions with a 75% deacetylated (DAc) chitosan suspension (pH 5.0) at room temperature. The Ag NPs were homogeneously dispersed and stably embedded in the chitosan matrices. The Ag NP/chitosan composites were obtained as yellow or brown flocs. It was estimated that approximately 60, 120 and 360 μg of the small, medium and large Ag NPs, respectively, were maximally embedded in 1 mg of chitosan. The bactericidal and anti-fungal activities of the Ag NP/chitosan composites increased as the amount of Ag NPs in the chitosan matrix increased. Furthermore, smaller Ag NPs (per weight) in the chitosan composites provided higher bactericidal and anti-fungal 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 (3.48±1.83 nm in diameter), medium (6.53±1.78 nm), and large (12.9±2.5 nm) particles, respectively. In this study, Ag NP/chitin composites were synthesized by mixing each of these three Ag NP suspensions with a 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.

Recent evidence indicates that low oxygen tension or hypoxia alters the characteristics of stem cells. The actions of hypoxia are mediated through the hypoxia-inducible factor, a critical mediator of the cellular response to hypoxia. Adipose tissue–derived stromal cells (ASCs) are one of the most promising cell sources for tissue engineering applications. This study investigated the effect of hypoxia on ASCs in terms of the ability to proliferate and differentiate. ASCs were extracted from mice and maintained under hypoxic atmosphere (2% O2) for up to eight in vitro passages. The proliferation rate was examined as a growth curve, and the potency of differentiation was evaluated. To investigate the cell characteristics, we checked several stem-cell markers and growth factors. Compared with the normoxic state (20% O2), hypoxia enhances proliferation with an approximately six- to sevenfold higher ASC expansion over 6 weeks. The expression of Oct3/4 and Nanog (stem-cell marker) and the amount of secreted growth factors were increased under the hypoxic condition. These results suggest that low oxygen tension enhances proliferation and maintains stemness of ASCs. Thus, this study emphasizes the profitability of hypoxic culture for expansion of ASCs and maintenance of their undifferentiated state for further therapeutic use.

Size-controlled spherical silver nanoparticles (

Yuki Takabayashi,1Masaki Nambu,1Masayuki Ishihara,2Masahiro Kuwabara,1Koichi Fukuda,2Shingo Nakamura,2Hidemi Hattori,2Tomoharu Kiyosawa1 1Department of Plastic and Reconstructive Surgery, 2Division of Biomedical Engineering, Research Institute, National Defense Medical College, Tokorozawa, Saitama, Japan Purpose: Although treatments for alopecia are in high demand, not all treatments are safe and reliable. Dalteparin/protamine nanoparticles (D/P NPs) can effectively carry growth factors (GFs) such as fibroblast GF (FGF)-2. The purpose of this study was to identify the effects of FGF-2-containing D/P NPs (FGF-2&D/P NPs) on hair growth.Patients and methods: In this study, the participants were 12 volunteers with thin hair. One milliliter of FGF-2 (100 ng/mL) and D/P NPs (56 μg/mL) was applied and massaged on the skin of the scalp by the participants twice a day. They were evaluated for 6months. Participants were photographed using a digital camera for general observation and a hair diagnosis system for measuring hair diameter.Results: The mean diameter of the hairs was significantly higher following the application of FGF-2&D/P NPs for 6months. Objective improvements in thin hair were observed in two cases. Nine participants experienced greater bounce and hair resilience.Conclusion: The transdermal application of FGF-2&D/P NPs to the scalp can be used as a new treatment for alopecia. Keywords: hair growth, dalteparin/protamine nanoparticles, fibroblast growth factor, transdermal application

Methotrexate (MTX) is used for the treatment of polyarticular juvenile idiopathic arthritis (JIA), and an anti-interleukin-6 receptor monoclonal antibody (tocilizumab: TCZ) is also used and added for the treatment of intractable JIA. It has been reported that MTX might induce Epstein-Barr virus (EBV)-associated lymphoma, but the discussion about the effect of MTX and/or TCZ against reactivation of EBV in pediatric patients has been incomplete.The EBV loads in four polyarticular JIA and three systemic arthritis JIA patients treated with MTX and/or TCZ, and the percentage of EBV-specific killer T cells (EBV-CTLs) in some patients were prospectively monitored.No patients had EBV-associated symptoms during the observation period. EBV loads in all patients were not significantly increased, and the levels of EBV loads were the same as EBV-seropositive healthy children following the administration of MTX and/or TCZ. EBV-CTLs were detectable during the observation period, but some patients had slightly low levels of EBV-CTLs.Treatment with MTX and/or TCZ did not severely affect EBV load and prevent induction of EBV-CTLs in JIA patients.

The effects of the optical properties of gold nanoparticle (AuNP) on the photoacoustic (PA) signals were investigated with the numerical simulation and experiment using various spherical and polyhedral AuNPs. In the numerical simulation, the absorption and scattering cross sections were calculated by the discrete dipole approximation. The absorbed light energy was calculated by the Monte Carlo method. Then PA pressure wave was simulated based on the PA wave equation. In the experiment, the PA signals from the AuNP suspensions were measured by use of a P(VDFTrFE) piezoelectric film and a Q-switched Nd:YAG laser operated at 532 nm. It was demonstrated that the PA signals were characterized by both of the absorption cross sections and the scattering cross section. The scattering medium containing the AuNPs enhanced the amplitude of the PA signals.

The purpose of the study was to evaluate the effects of isogenous platelet-rich plasma (PRP)–containing fragmin/protamine microparticles (F/P MPs) as a delivery system for proteins in PRP on growth of endothelial and smooth muscle cells (SMCs) in vitro and as an alternative treatment for peripheral arterial disease (PAD) and critical limb ischemia. Frozen and thawed PRP contains high concentrations of growth factors that are adsorbed by F/P MPs. Human aorta endothelial cells (AECs) and SMCs were grown in a medium with PRP. Addition of F/P MPs significantly enhanced the proliferative effects of PRP on AECs and SMCs at 37°C for >10 days. Intramuscular administration of phosphate-buffered saline (PBS; 2 mL, control), F/P MPs (12 mg in 2 mL PBS), PRP (2 mL), or PRP (2 mL) containing F/P MPs (12 mg) was then performed in a rabbit model of hindlimb ischemia prepared by resection of the left femoral artery. Blood flow and pressure were measured on days 0, 14, and 28, and angiography to assess arteriogenesis was performed on day 28. PRP-containing F/P MPs strongly induced functional collateral vessels in the rabbit model of hindlimb ischemia, indicating possible use of these microparticles in therapy for PAD. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 36–42, 2013.

The use of liposome-encapsulated hemoglobin (LHb), which is a cellular Hb, has been demonstrated to be beneficial in the treatment of hypohemoglobinemic shock. As a molecule of appropriate size (220 nm) that can carry oxygen, LHb may ameliorate cardiac dysfunction during lethal hemodilation. The purpose of this study was to determine the efficacy of LHb transfusion in relieving cardiovascular dysfunction in a rat model of lethal progressive hemodilution. Over the course of 150 min, rats were subjected to blood withdrawal (0.2 mL/min) and simultaneously transfused with LHb, washed rat red blood cells, or 5% albumin. Temporal changes in cardiac function, heart-type fatty acid-binding protein levels, plasma levels of catecholamines, heart rate variability, and hypoxia-inducible factor 1α expression were measured during lethal progressive hemodilution. More than 80% of the rats transfused with either LHb or washed rat red blood cells survived for 8 days. Liposome-encapsulated hemoglobin transfusion suppressed hypoxia-inducible factor 1α expression in the heart, maintained low levels of heart-type fatty acid-binding protein, and attenuated sympathetic nerve activity as reflected by changes in heart rate variability and plasma levels of epinephrine and norepinephrine. The results indicate that LHb attenuates cardiac dysfunction and sympathetic overactivity during lethal hemorrhage.

This article is concerned with the theoretical analysis of the functionally graded magneto-electro-thermoelastic hollow cylinder due to uniform surface heating. We analyze the transient thermal stress problem for a functionally graded hollow cylinder constructed of the anisotropic and linear magneto-electro-thermoelastic materials using a laminated composite model as one of theoretical approximation under a plane strain state. As an illustration, we carry out numerical calculations for a functionally graded hollow cylinder constructed of piezoelectric and magnetostrictive materials and examined the behaviors in the transient state. We investigate the effects of the nonhomogeneity of material on the stresses, electric potential, and magnetic potential, and the effect of the applied electric potential on the thermal stress σθθ.

This paper presents the theoretical analysis of a multilayered magneto-electro-thermoelastic hollow sphere under unsteady and uniform surface heating. We obtain the exact solution of the transient thermal stress problem of the multilayered magneto-electro-thermoelastic hollow sphere in the spherically symmetric state. As an illustration, we perform numerical calculations of a two-layered composite hollow sphere made of piezoelectric and magnetostrictive materials and investigate the numerical results for temperature change, displacement, stress, and electric and magnetic potential distributions in the transient state are shown in figures.

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.

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 © 2012 John Wiley & Sons, Ltd.

In the field of orthopaedic surgery, an orthopaedic surgeon sometimes requires to suppress excessive bone formation, such as ectopic bone formation, ossifying myositis and radio-ulnar synostosis, etc. Ultraviolet (UV) light irradiation of a photocrosslinkable chitosan (Az-CH-LA) generates an insoluble hydrogel within 30 s. The purpose of this study was to evaluate the ability of the photocrosslinked chitosan hydrogel (PCH) to inhibit bone formation in an experimental model of bone defect. Rat calvarium and fibula were surgically injured and PCH was implanted into the resultant bone defects. The PCH implants significantly prevented bone formation in the bone defects during the 4 and 8 week observation periods. In the PCH-treated defects, fibrous tissues infiltrated by inflammatory cells were formed by day 7, completely filling the bone defects. In addition to these findings, expression of osteocalcin and runt-related gene 2 (RUNX2) mRNA, both markers of bone formation, was lower in the PCH-treated defects than in the controls. In contrast, collagen type 1α2 and α-smooth muscle actin (α-SMA) mRNA levels were significantly higher in the PCH-treated defects after 1 week. PCH stimulated the formation of fibrous tissue in bone defects while inhibiting bone formation. Thus, PCH might be a promising new therapeutic biomaterial for the prevention of bone formation in orthopaedic surgery.

We examined the effects of prostaglandin E₁ (PGE₁) on radiation-induced proliferation inhibition and apoptosis in keratinocytes and healing of radiation-induced skin injury in a rat model. PGE₁ had a protective effect on radiation-induced growth inhibition in keratinocytes in vitro, but not in fibroblasts. Varying concentrations of PGE₁ were subcutaneously administered into the posterior neck region. X-irradiation at a dose of 20 Gy was administrated to the lower part of the back using a lead sheet with two holes 30 min to 1 h before or after the administration of PGE₁. Although X-irradiation induced epilation, minor erosions, or skin ulcers in almost all rats, PGE₁ administration prior to irradiation reduced these irradiation injuries. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling showed that proportions of apoptotic keratinocytes in the X-irradiated skin of PGE₁-administered rats were significantly lower than for those in the skin of rats which did not receive PGE₁. Cutaneous full-thickness defective wounds were then formed in X-irradiated areas to examine the time course of wound healing. Wound healing was significantly delayed because of X-irradiation, but PGE₁ administration prior to irradiation led to a significantly shorter delay in wound healing compared with controls. Decreasing delay in wound healing was correlated with concentration of PGE₁ administrated. Thus, PGE₁-administration may potentially alleviate the radiation-induced skin injury.

Low-molecular-weight heparin/protamine microparticles (LMW-H/P MPs) were produced as a carrier for heparin-binding growth factors (GFs) and for various adhesive cells. A mixture of low-molecular-weight heparin (MW: approximately 5000 Da, 6.4 mg/mL) and protamine (MW: approximately 3000 Da, 10 mg/mL) at a ratio of 7:3 (vol:vol) yields a dispersion of microparticles (0.5–3 µm in diameter). LMW-H/P MPs immobilize, control the release and protect the activity of GFs. LMW-H/P MPs can also bind to cell surfaces, causing these cells to interact with the LMW-H/P MPs, inducing cells/MPs-aggregate formation and substantially promoting cellular viability. Furthermore, LMW-H/P MPs can efficiently bind to tissue culture plates and retain the binding of important GFs, such as fibroblast growth factor (FGF)-2. The LMW-H/P MPs-coated matrix with various GFs or cytokines may provide novel biomaterials that can control cellular activity such as growth and differentiation. Thus, LMW-H/P MPs are an excellent carrier for GFs and various cells and are an efficient coating matrix for cell cultures.

Fragmin/protamine nanoparticles (F/P NPs) have been used as carriers for the preservation and controlled release of fibroblast growth factor (FGF)-2 and various cytokines in human plasma (HP). This study tested an HP–Dulbecco's modified Eagle's medium (DMEM) gel as a three-dimensional (3D) culture for the expansion of adipose tissue-derived multilineage stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs). The growth of these cells improved in 3D culture using low-concentration HP (2%)–DMEM gel with 0.1 mg/mL F/P NPs and 5 ng/mL FGF-2 without animal serum in comparison to two-dimensional (2D) culture using a low-concentration human serum (2%)–DMEM containing 5 ng/mL FGF-2 on F/P NPs-coated plates. ASCs and BMSCs, which were expanded in the low-concentration HP–DMEM gel with F/P NPs and FGF-2, maintained their multilineage potential for differentiation into adipocytes or osteoblasts similar to the 2D cultured cells. Furthermore, flow cytometric analyses showed that the phenotypic markers which were positive for CD44, CD90, and CD105 (>80%) and negative for CD34 and CD45 (

The optimization problem for tensioning conditions in a rotating circular saw under a thermal load is solved by a genetic algorithm. The governing equations for the in-plane behavior due to rotation, thermal load by friction, and plastic strain by tensioning and those for the resulting out-of-plane behavior are presented; the solution for in-plane forces is obtained; and a modal analysis is performed. Numerical calculations are performed to investigate the effect of tensioning conditions on the natural frequencies. The optimization problem to maximize the natural frequency of the most critical mode is solved, and the optimal tensioning conditions are determined.

This article is concerned with the theoretical analysis of the functionally graded magneto-electro-thermoelastic hollow sphere due to uniform surface heating. We analyze the transient thermoelastic problem for a functionally graded hollow sphere constructed of the spherical isotropic and linear magneto-electro-thermoelastic materials using a laminated composite mode as one of theoretical approximation in the spherically symmetric state. As an illustration, we carry out numerical calculations for a functionally graded hollow sphere constructed of piezoelectric and magnetostrictive materials and examine the behaviors in the transient state. The effects of the nonhomogeneity of material on the stresses, electric potential, and magnetic potential are investigated.

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.

ObjectivesThe localized delivery of exogenous, angiogenic growth factors such as fibroblast growth factor (FGF)-2 has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia (CLI). The present study describes the efficacy of fragmin/protamine microparticles containing FGF-2 (F/P-MPs/FGF-2) to promote vessel growth in a rabbit model of hindlimb ischemia.MethodsA total of 24 rabbits were used to construct a model of hindlimb ischemia by resection of the left femoral artery. The rabbits were randomly divided into four groups 10 days after surgery (day 0); group A: control (non-treated; 1 mL of phosphate-buffered saline [PBS]); group B: FGF-2 (100 μg FGF-2 in 1 mL PBS)-treated; group C: F/P-MPs (12 mg dried F/P MPs in 1 mL PBS)-treated; and group D; F/P MPs/FGF-2 (100 μg FGF-2 and 12 mg dried F/P MPs in 1 mL PBS)-treated (n = 6 each). The drugs were administered intramuscularly to each group. Blood flow and blood pressure were measured in each group on days 0, 14, and 28. Angiography was performed to assess arteriogenesis on day 28. The number of capillaries on day 28 was determined by direct counting CD31− and α-smooth muscle antibody (α-SMA)-positive vessels.ResultsNeither death nor wound infection was observed throughout the experiment. The F/P MPs/FGF-2-treated group showed marked improvement in the blood flow ratio, blood pressure ratio, and capillary number in comparison to the control group, FGF-2-treated group, and F/P MPs-treated group. The F/P MPs-treated group showed intermediate improvement in blood flow ratio and capillary number in comparison to the control group and FGF-2-treated group.ConclusionsThe F/P MPs/FGF-2-treated group strongly induced functional collateral vessels in the rabbit model of hindlimb ischemia, indicating a possible therapy for PAD.Clinical RelevancePAD due to atherosclerotic vascular disease is a major health problem. Despite recent advances in surgical and radiologic vascular techniques, certain patients with CLI are not suitable for revascularization. A variety of strategies have been tried to promote development of collateral vessels. F/P MPs can act as carriers for controlled release of FGF-2. The purpose of this study was to evaluate the efficacy of F/P MPs/FGF-2 to induce functional collateral vessels in a rabbit model of hindlimb ischemia. This study will lead to F/P MPs/FGF-2-therapy which is an effective therapeutic strategy for treating PAD patients in clinic.

This article is concerned with the theoretical analysis of the functionally graded magneto-electro-thermoelastic strip due to unsteady and nonuniform surface heating in the width direction. We analyze the transient thermal stress problem for a functionally graded strip constructed of the anisotropic and linear magneto-electro-thermoelastic materials using a laminated composite mode as one of theoretical approximation. The transient two-dimensional temperature is analyzed by the methods of Laplace and finite sine transformations. We obtain the solution for the simply supported and functionally graded magneto-electro-thermoelastic strip under a plane strain state. As an illustration, we carried out numerical calculations for a functionally graded strip composed of piezoelectric BaTiO3 and magnetostrictive CoFe2O4, and examined the behaviors in the transient state for temperature change, stress, electric potential and magnetic potential distributions. Furthermore, the effects of the nonhomogeneity of material on the stresses, electric potential, and magnetic potential are investigated.

Mixing a low-molecular-weight heparin (e.g., fragmin) with protamine results in the formation of water-insoluble fragmin/protamine microparticles (F/P MPs) approximately 0.5–3 μm in diameter. In this study, we investigated the ability of F/P MPs to improve the viability of Lewis lung (3LL) cancer cells, B16 (B16) melanoma cells, and a human hepatoma (Huh7) cell line in a suspension culture, and the ability of F/P MPs to enhance tumor take rate and growth in vivo. F/P MPs rapidly bound to the surface of the cells. The interaction of the cells with F/P MPs induced formations of the tumor cell/F/P MP aggregates and maintained the viability of those cells in suspension for at least 3 days. The addition of F/P MPs with FGF-2 significantly enhanced the growth rate of the cells in a fetal bovine serum (FBS)-free medium. The tumor cell/F/P MP aggregates, which were subcutaneously injected into the backs of mice, significantly stimulated the formation and growth of subcutaneous tumors consisting of 3LL, B16, and Huh7 cells. Furthermore, the tumors produced by injection of 7.5 × 105 Huh7 into nude mice did grow only with F/P MPs. Thus F/P MPs can utilize as cell carrier for tumor cell transplantation.

Tanaka E, Sato T, Ishihara M, Tsutsumi Y, Hisano M, Chikamoto H, Akioka Y, Dohno S, Maeda A, Hattori M, Wakiguchi H, Fujieda M. Asymptomatic high Epstein-Barr viral load carriage in pediatric renal transplant recipients. Pediatr Transplantation 2011: 15: 306–313. © 2011 John Wiley & Sons A/S. Abstract: High viral load carriage of EBV is one of the risks for PTLD in transplant recipients. We reviewed retrospectively in pediatric renal transplant recipients with EBV seronegative. EBV loads in peripheral blood and EBV-CTLs were measured every 1–3 months in 13 patients after grafting. Immunosuppressants were reduced when the patients were considered to have persistent high EBV loads (>1000 copies/μgDNA for over six months). All showed primary EBV infection: six with asymptomatic persistent high EBV loads (group A) and seven with neither EBV-associated symptoms nor persistent high EBV loads (group B). No patient developed PTLD in either group. Chronic rejection occurred in one patient in group A after immunosuppressants’ reduction. There was no difference in renal dysfunction rates between the two groups. The maximum and increase rates in EBV loads were significantly higher in group A. The CTLs’ percentage was significantly lower in group A when EBV loads first rose above 100 copies/μg DNA. This study suggests the possibility that EBV loads and CTLs’ monitoring may be useful for avoidance of PTLD, as patients with asymptomatic persistent high EBV loads had higher EBV loads and lower percentages of CTLs.

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.

Recently, we developed liposome-encapsulated hemoglobin (LEH), a novel cellular hemoglobin-based oxygen carrier. We hypothesized that the LEH effectively suppresses scavenging of nitrogen oxides by sequestering hemoglobin, thereby being useful for resuscitation from hemorrhagic shock, especially in prehospital settings where blood transfusion is not available. However, putting a catheter into the peripheral vessels is sometimes difficult in prehospital resuscitation, because these vessels collapse in patients with hemorrhagic shock. The intraosseous route does not collapse under such conditions. We here studied the resuscitation of severe hypohemoglobinemia following massive hemorrhage using intraosseous (intrafemur) transfusion with LEH in mice. First, we examined the effect of intravenous transfusion with LEH on the resuscitation of mice with fatal hypohemoglobinemia that was made with progressive hemodilution by blood exchanges. Despite a success in initial resuscitation without scavenging of NO2 or NO3, LEH transfusion did not significantly improve mouse survival 72 h later as compared with red blood cell (RBC) transfusion. In other experiments, hypohemoglobinemic mice were also made with blood withdrawal and intraosseous infusion with 5% albumin. Thereafter, the mice were rescued with intraosseous transfusion of LEH or RBCs. Unlike intravenous transfusion, intraosseous transfusion with LEH (but not such transfusion with RBCs) significantly increased mouse survival without scavenging of NO2 or NO3, presumably because LEH vesicles were much smaller than RBCs, thereby effectively flowing into the circulation from the femur. Thus, intraosseous transfusion with LEH may be a candidate strategy for efficient prehospital resuscitation from hemorrhagic shock.

We produced low-molecular-weight heparin/protamine micro (nano) particles (LMW-H/P MPs·NPs) as a carrier for heparin-binding growth factors (GFs), such as fibroblast growth factor (FGF)-2 and various GFs in platelet-rich plasma (PRP). A mixture of LMW-H (MW: approximately 5000 Da, 6.4 mg/ml) and protamine (MW: approximately 3000 Da, 10 mg/ml) at a ratio of 7:3 (vol:vol) yields a dispersion of micro (nano) particles (200 nm - 3 µm in diameter). The diluted LMW-H 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 (approximately 100 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 NPs solution. The LMW-H/P MPs·NPs adsorb GFs, control their release, protect GFs and activate their biological activities. Furthermore, administration of GFs-containing F/P MPs·NPs exhibited significantly higher inductions of vascularization and fibrous tissue formation in vivo than GFs alone. LMW-H/P MPs·NPs can also efficiently bind to tissue culture plates and retain the binding of GFs. The LMW-H/P MPs·NP-coated matrix with various GFs or cytokines provided novel biomaterials that could control cellular activity such as proliferation and differentiation. Thus, LMW-H/P MPs·NPs are an excellent carrier for GFs and are a functional coating matrix for various kinds of cell cultures.

To lighten the workload of health care professionals, we conducted a clinical test of a newly developed automated infection screening system using a multivariable logistic regression model. The system was tested with 44 influenza patients and 45 healthy control subjects based on 3 vital signs: facial temperature, heart rate and respiratory rate. The system showed a high accuracy for distinguishing influenza patients from control subjects within 15 seconds.

A simple, environmentally friendly method for preparing highly size-controlled spherical silver nanoparticles was developed that involved heating a mixture of silver-containing glass powder and an aqueous solution of glucose. The stabilizing agent for silver nanoparticles was found to be caramel, which was generated from glucose when preparing the nanoparticles. The particle size was independent of the reaction time, but it increased proportionally with the square root of the glucose concentration in the range 0.25–8.0 wt% (corresponding to particle sizes of 3.48 ± 1.83 to 20.0 ± 2.76 nm). Difference of the generation mechanism of silver nanoparticles between this inhomogeneous system and a system in which Ag+ was homogeneously dispersed was discussed.

Hydrodynamics-based gene delivery (HGD) is an efficient method for transfecting plasmid DNA into hepatocytes in vivo. However, the resulting gene expression is transient, and occurs in a non-tissue specific manner. The piggyBac (PB) transposon system allows chromosomal integration of a transgene in vitro. This study aimed to achieve long-term in vivo expression of a transgene by performing hepatocyte-specific chromosomal integration of the transgene using PB and HGD. Using this approach, we generated a novel mouse model for a hepatic disorder. A distinct signal from the reporter plasmid DNA was discernible in the murine liver approximately two months after the administration of PB transposons carrying a reporter gene. Then, to induce the hepatic disorder, we first administered mice with a PB transposon carrying a CETD unit (loxP-flanked stop cassette, diphtheria toxin-A chain gene, and poly(A) sites), and then with a plasmid expressing the Cre recombinase under the control of a liver-specific promoter. We showed that this system can be used for in situ manipulation and analysis of hepatocyte function in vivo in non-transgenic (Tg) animals.

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.

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.

We investigated whether pravastatin ameliorates renal damage induced by cisplatin (CP). Forty-three male Wistar rats were divided into four groups: rats treated with a control diet for 19 days and saline injection on day 14 (group1), group 1 with pravastatin treatment with 19 days (group 2), group 1 with CP injection on day 14 (group 3), and group 2 with CP injection (group 4). Renal function and serum lipids, renal malondialdehyde (MDA) and glutathione (GSH) levels, glutathione peroxidase (GPx) mRNA expression and activity, and kidney triglyceride (TG) concentrations were measured. Histology was evaluated by light microscopy with immunohistochemistry for p53, p53-upregulated modulation of apoptosis (PUMA), and terminal deoxynucleotide transferase dUTP nick end-labeling (TUNEL) staining. CP induced renal tubular damage with a higher MDA level, increased PUMA expression, p53- and TUNEL-positive cells counts, elevation of serum lipids, and decreased GSH level, GPx mRNA expression, and activity. Pravastatin partially ameliorated CP-induced renal injury, based on suppression of the renal MDA and TG levels, decreased p53 expression, and apoptosis in CP-treated rats. These findings suggest that pravastatin has a partial protective effect against CP nephrotoxicity via antioxidant activity as well as attenuation of the p53 response, and lipid-lowering effects.

This study aimed to examine the relationships among electroencephalogram parameters as physiological indices, ballistic movement as an index of operation, and accuracy of task performance. Experiments were conducted using a “reaching” task, in which subjects touched the target appearing 300 pixels away from a start point in a vertical direction on a touch-sensitive screen with the forefinger. During experiments, EEG, EMG as trigger, high-speed camera images, and task efficiency were acquired. Significant differences between the high- and low-performance groups were clearly confirmed on the slope of NS (negative slope) in movement-related cortical potentials (MRCPs) acquired from Fz (p < 0.05) and Cz (p < 0.05). Furthermore, a difference between these groups was confirmed in duration of ballistic movement. Based on our findings, we discuss whether it is possible to extract MRCP rapidly and automatically without using signal averaging and also to estimate accuracy just before a motion is executed.

In patients with severe hemorrhage, complications such as shock or death may occur if the patient is not treated appropriately and expeditiously. To create a hemostat kit for severe hemorrhage, ultraviolet light irradiation was applied to photocrosslinkable chitosan hydrogel and calcium alginate. As a hemorrhage model, the femoral arteries and veins of anesthetized rats were cut. Hemodynamics and hematological parameters including red blood cell (RBC) count, hemoglobin concentration, hematocrit, white blood cell (WBC) count, and platelet count, and serum parameters including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as a marker of hemostasis. In rats for which no procedure was used, death occurred within 30 min. By using the hydrogel hemostat, the survival rate rose to 75% or more. RBC count, hemoglobin, hematocrit, and platelet levels were not significantly changed for 3 days. WBC count increased 1 day after hemostasis. AST and ALT increased 1 day after hemostasis, but it decreased 3 days later. The photocrosslinkable chitosan hydrogel and calcium alginate were biodegraded at 3 and 28 days, respectively, by neutrophils and keratinocyte chemoattractant.

To convert cellulosic organics contained in industrial paper sludge into glucose, reaction of pseudo paper sludge composed of cellulose and inorganic compounds (calcium carbonate (CaCO 3 ), talc (Mg 3 (Si 4 O 10 )(OH) 2 ), kaolin (Al 2 (Si 2 O 5 )(OH) 4 )) under hydrothermal conditions was studied. Significant amounts of glucose ( ca. 23%) could be produced from cellulose in the absence of CaCO 3 for reaction in water at 523 K and 12 min reaction time. On the other hand, in the presence of CaCO3, most glucose decomposed over all conditions, whereas the addition of talc and kaolin to the mixtures increased the glucose yield to about 30%. For the case of chemical recycle of paper sludge with hydrothermal treatment to obtain d -glucose, it can be concluded that it is preferable to separate the calcium carbonate from the paper sludge before hydrothermal treatment.

Many compensatory mechanisms exit in hemorrhagic shock (HS). To characterize the efficacy of the new artificial oxygen carrier, liposome-encapsulated hemoglobin (LHb), HS was induced by withdrawing 20% of the total blood volume from rats. Rats received one of five interventions: LHb resuscitation (LHb-G, n = 7), normal saline (Saline-G, n = 7), shed autologous blood (SAB-G, n = 7), volume expander of 5% albumin (Albumin-G, n = 7), or no treatment (Sham-G, n = 7). Heart rate variability (HRV) indices were measured, including low frequency (LF, 0.10–0.60 Hz), high frequency (HF, 0.60–2.00 Hz), and the ratio of LF to HF (LF/HF). LF and LF/HF following HS were lower in the LHb-G and SAB-G groups when compared with the Saline-G, Albumin-G and Sham-G groups. LF and LF/HF following HS in the LHb-G group were comparable with that of the SAB-G group. These data demonstrate that HS-induced changes can be attenuated by resuscitation with LHb as well as SAB. LHb could be used as a substitute for blood transfusion for HS.

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.