Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells.

BACKGROUND: We evaluated the efficacy of the factor Xa inhibitor rivaroxaban on the differentiation ability of vascular endothelial progenitor cells (EPCs), which play roles in vascular injury repair and atherogenesis. Antithrombotic treatment in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI) is challenging, and current guidelines recommend oral anticoagulant monotherapy 1 year or more after PCI. However, biological evidence of the pharmacological effects of anticoagulants is insufficient. METHODS: EPC colony-forming assays were performed using peripheral blood-derived CD34-positive cells from healthy volunteers. Adhesion and tube formation of cultured EPCs were assessed in human umbilical cord-derived CD34-positive cells. Endothelial cell surface markers were assessed using flow cytometry, and Akt and endothelial nitric oxide synthase (eNOS) phosphorylation were examined using western blot analysis of EPCs. Adhesion, tube formation and endothelial cell surface marker expression was observed in EPCs transfected with small interfering RNA (siRNA) against protease-activated receptor (PAR)-2. Finally, EPC behaviors were assessed in patients with atrial fibrillation undergoing PCI in whom warfarin was changed to rivaroxaban. RESULTS: Rivaroxaban increased the number of large EPC colonies and increased the bioactivities of EPCs, including adhesion and tube formation. Rivaroxaban also increased vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, Tie-2, and E-selectin expression as well as Akt and eNOS phosphorylation. PAR-2 knockdown increased the bioactivities of EPCs and endothelial cell surface marker expression. Patients in whom the number of large colonies increased after switching to rivaroxaban showed better vascular repair. CONCLUSIONS: Rivaroxaban increased the differentiation ability of EPCs, leading to potential advantages in the treatment of coronary artery disease.

GATA6 regulates expression of annexin A10 (ANXA10) associated with epithelial-mesenchymal transition of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) can disturb oral function and quality of life and is associated with poor survival, likely due to the development of cervical lymph node metastases. Epithelial-mesenchymal transition (EMT) is a process in which cells acquire molecular alterations that facilitate cell motility and invasion, and has been associated with tumor metastasis. EMT changes also play important roles in the induction of lymph node metastasis in OSCC. GATA6 is known as the earliest marker of the primitive endoderm lineages. GATA6 inhibits de-differentiation and EMT in human pancreatic ductal adenocarcinoma cells and promotes EMT. However, in OSCC, the expression and function of GATA6 in EMT and lymph node metastasis remains unclear. Therefore, this study aimed to clarify the targets of GATA6 in OSCC cells and whether the change in GATA6 expression affects EMT in OSCC cells, as well as the association between GATA6 and lymph node metastasis. The results showed that GATA6 knockdown OSCC cells promoted EMT and increased lymph node metastasis compared with control cells, whereas the overexpression of GATA6 inhibited the induction of EMT and reduced lymph node metastasis. In addition, annexin A10 (ANXA10) which is the largest type of Ca2+-regulated phospholipid-binding protein in eukaryotic cells was detected as a target gene for GATA6 and ANXA10 suppressed Vimentin expression in EMT in OSCC. Therefore, the GATA6/ANXA10 cascade may be a potential therapeutic approach for the treatment of lymph node metastases in OSCC patients.

Local transplantation of syngeneic adipose stromal vascular fraction ameliorates damaged anal sphincter function in a rat model of vaginal distension.

BACKGROUND: The adipose stromal vascular fraction contains abundant mesenchymal stem cells and is utilized for cell therapy of male stress urinary incontinence. The purpose of this paper was to explore the effect of local transplantation of the stromal vascular fraction on improvement of damaged anal sphincter function. METHODS: A rat model of vaginal distension was used as a model of damaged anal sphincter function. The adipose stromal vascular fraction was separated from the inguinal fat of syngeneic green fluorescent protein transgenic rats and delivered into the internal anal sphincter of vaginal distension rats. The maximum resting pressure was evaluated during insertion and withdrawal of the catheter at 4 or 10 days after vaginal distension treatment to estimate anal sphincter function. Green fluorescent protein-transfected human-adipose-derived mesenchymal stem cells were transplanted into the internal anal sphincter of nude rats. Hematoxylin-eosin and Masson trichrome staining were performed to evaluate tissue damage and collagen synthesis. Transplanted cells were identified using a green fluorescent protein antibody and a human-specific antibody. Activation of the transplanted human-ADSC was evaluated by quantitative RT-PCR RESULTS: The mean maximum resting pressure (during catheter withdrawal) of vaginal distension rats was significantly lower than that of control rats, and stromal vascular fraction injection normalized it 4 days after treatment (control: 5.66 ± 0.98, vaginal distension: 4.04 ± 1.28, vaginal distension + stromal vascular fraction: 5.92 ± 1.28 [mmHg, control versus vaginal distension: P = .039; vaginal distension versus vaginal distension + stromal vascular fraction: P = .007]). Histological examination showed that vaginal distension disrupted the internal anal sphincter, and the transplanted syngeneic stromal vascular fraction survived for 10 days. Transplanted xenogeneic human-adipose-derived mesenchymal stem cells survived in the internal anal sphincter of nude rats for 4 and 10 days. Genes related to extracellular remodeling were up-regulated in the transplanted human-adipose-derived mesenchymal stem cells CONCLUSION: Syngeneic and heterotopic transplanted adipose-derived mesenchymal stem cells engrafted in the internal anal sphincter and ameliorated damaged anal sphincter function in a rat model of vaginal distension.

Surgical Injury and Ischemia Prime the Adipose Stromal Vascular Fraction and Increase Angiogenic Capacity in a Mouse Limb Ischemia Model.

The adipose-derived stromal vascular fraction (SVF) is an effective source for autologous cell transplantation. However, the quality and quantity of SVFs vary depending on the patient's age, complications, and other factors. In this study, we developed a method to reproducibly increase the cell number and improve the quality of adipose-derived SVFs by surgical procedures, which we term "wound repair priming." Subcutaneous fat from the inguinal region of BALB/c mice was surgically processed (primed) by mincing adipose parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). SVFs were isolated on day 0, 1, 3, 5, or 7 after the priming procedures. Gene expression levels of the primed SVFs were measured via microarray and pathway analyses which were performed for differentially expressed genes. Changes in cellular compositions of primed SVFs were analyzed by flow cytometry. SVFs were transplanted into syngeneic ischemic hindlimbs to measure their angiogenic and regeneration potential. Hindlimb blood flow was measured using a laser Doppler blood perfusion imager, and capillary density was quantified by CD31 staining of ischemic tissues. Stabilization of HIF-1 alpha and VEGF-A synthesis in the SVFs were measured by fluorescent immunostaining and Western blotting, respectively. As a result, the number of SVFs per fat weight was increased significantly on day 7 after priming. Among the differentially expressed genes were innate immunity-related signals on both days 1 and 3 after priming. In primed SVFs, the CD45-positive blood mononuclear cell fraction decreased, and the CD31-CD45-double negative mesenchymal cell fraction increased on day 7. The F4/80-positive macrophage fraction was increased on days 1 and 7 after priming. There was a serial decrease in the mesenchymal-gated CD34-positive adipose progenitor fraction and mesenchymal-gated CD140A-positive/CD9-positive preadipocyte fraction on days 1 and 3. Transplantation of primed SVFs resulted in increased capillary density and augmented blood flow, improving regeneration of the ischemic limbs. HIF-1 alpha was stabilized in the primed cutaneous fat in situ, and VEGF-A synthesis of the primed SVFs was on a peak on 5 days after priming. Wound repair priming thus resulted in SVFs with increased number and augmented angiogenic potential.

Cancer-associated fibroblasts show heterogeneous gene expression and induce vascular endothelial growth factor A (VEGFA) in response to environmental stimuli.

AIM: Cancer-associated fibroblasts (CAF) play a crucial role in angiogenesis in the complex tumor microenvironment. However, fibroblasts show extensive heterogeneity and their dynamic functions against stressors remain largely unknown. METHODS: We collected patient-derived CAF and carried out perturbation-based monitoring of the dynamic functions. Clinically relevant experimental stimuli were defined as follows: hypoxia, cisplatin, fluorouracil, coculture with cancer spheroids (interaction through paracrine signals). We selected 18 marker genes that encode components for fibroblast activation, intracellular communication, and extracellular matrix remodeling. Quantitative reverse transcription polymerase chain reaction was carried out for data collection and statistical analyses were carried out using SPSS software. RESULTS: Kruskal-Wallis multivariate analysis of variance showed that variations in expression of 11 marker genes were explained, in part, by a difference in tissue of origin. Friedman and two-sided Wilcoxon signed rank tests detected significant perturbations in expression of marker genes. Paracrine signal from cancer spheroids induced vascular endothelial growth factor A (VEGFA) in CAF but not in fetal lung fibroblasts. CONCLUSION: We have established perturbation-based monitoring of patients' CAF. Further data collection and individual patient follow up is ongoing to identify critical determinants of disease outcome.

Polyelectrolyte Complexes of Natural Polymers and Their Biomedical Applications.

Polyelectrolyte complexes (PECs), composed of natural and biodegradable polymers, (such as positively charged chitosan or protamine and negatively charged glycosaminoglycans (GAGs)) have attracted attention as hydrogels, films, hydrocolloids, and nano-/micro-particles (N/MPs) for biomedical applications. This is due to their biocompatibility and biological activities. These PECs have been used as drug and cell delivery carriers, hemostats, wound dressings, tissue adhesives, and scaffolds for tissue engineering. In addition to their comprehensive review, this review describes our original studies and provides an overview of the characteristics of chitosan-based hydrogel, including photo-cross-linkable chitosan hydrogel and hydrocolloidal PECs, as well as molecular-weight heparin (LH)/positively charged protamine (P) N/MPs. These are generated by electrostatic interactions between negatively charged LH and positively charged P together with their potential biomedical applications.

Three-dimensional cultured tissue constructs that imitate human living tissue organization for analysis of tumor cell invasion.

Preventing cancer metastasis requires a thorough understanding of cancer cell invasion. These phenomena occur in human 3-D living tissues. To this end, we developed a human cell-based three-dimensional (3-D) cultured tissue constructs that imitate in vivo human tissue organization. We investigated whether our 3-D cell culture system can be used to analyze the invasion of human oral squamous cell carcinoma (OSCC) cells. The 3-D tissue structure consisted of five layers of normal human dermal fibroblasts along with human dermal lymphatic endothelial cell tubes and was generated by the cell accumulation technique and layer-by-layer assembly using fibronectin and gelatin. OSCC cells with different lymph metastatic capacity were inoculated on the 3-D tissues and their invasion through the 3-D tissue structure was observed. Conventional methods of analyzing cell migration and invasion, that is, 2-D culture-based transwell and Matrigel assays were also used for comparison. The results using the 3-D cultured tissue constructs were comparable to those obtained using conventional assays; moreover, use of the 3-D system enabled visualization of differential invasion capacities of cancer cells. These results indicate that our 3-D cultured tissue constructs can be a useful tool for analysis of cancer cell invasion in a setting that reflects the in vivo tissue organization. © 2018 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 292-300, 2019.

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.

Autologous and heterotopic transplantation of adipose stromal vascular fraction ameliorates stress urinary incontinence in rats with simulated childbirth trauma.

INTRODUCTION: Autologous transplantation of adipose stromal vascular fraction (SVF) is a cost-effective and technically accessible option for cell therapy. Clinical study of SVF transplantation for male stress urinary incontinence (SUI) is underway, but the effectiveness remains unknown for female SUI, majority of which is caused by childbirth trauma. METHODS: Vaginal Distension (VD) rats were generated as in vivo model for female SUI. To quantitate the severity of SUI, leak point pressure (LPP) was measured by placing a bladder catheter. There was a characteristic waveform of LPP with two-peaks, and we counted the second peak as an LPP value. Adipose SVF was separated from inguinal fat and delivered into external urethral sphincter (EUS) through transperineal injection. LPP was measured 7 or 14 days after SVF transplantation. Tissue damage and collagen synthesis around the EUS were visualized by Masson's trichrome and eosin staining. Antibody against α-smooth muscle actin (α-SMA) was used to stain smooth muscle or activated stromal cells. Donor SVF cells were distinguished from recipient EUS tissue by tracking with GFP transgene. RESULTS: VD procedure decreased the frequency at which the normal LPP waveform appeared and lowered the LPP value. SVF injection normalized the waveform as well as the level of LPP. VD disrupted histological structure of EUS and SVF failed to differentiate into striatal muscles. Instead, SVF increased α-SMA positive cells and collagen synthesis but the phenomena depended on VD stimulus. GFP tracking indicated that the transplanted SVF cells persisted for four weeks and synthesized α-SMA protein simultaneously. CONCLUSIONS: Autologous transplantation of adipose SVF displayed bulking effects through collagen synthesis. However, such heterotopic activation was dependent on tissue damage.

In vitro 3D blood/lymph-vascularized human stromal tissues for preclinical assays of cancer metastasis.

Tumour models mimicking in vivo three-dimensional (3D) microenvironments are of increasing interest in drug discovery because of the limitations inherent to current models. For example, preclinical assays that rely on monolayer or spheroid cell cultures cannot easily predict 3D cancer behaviours because they have no vasculature. Furthermore, there are major differences in cancer behaviour between human and animal experiments. Here, we show the construction of 3D blood/lymph-vascularized human stromal tissues that can be combined with cancer cells to mimic dynamic metastasis for real-time throughput screening of secreted proteinases. We validated this tool using three human carcinoma cell types that are known to invade blood/lymph vessels and promote angiogenesis. These cell types exhibited characteristic haematogenous/lymphogenous metastasis and tumour angiogenesis properties. Importantly, these carcinoma cells selectively secreted different matrix metalloproteinases depending on their metastasis stage and target vasculature, suggesting the possibility of developing drugs that can target each secreted proteinase. We conclude that the 3D tissue tool will be a powerful throughput system for predicting cancer cell responses and time-dependent secretion of molecules in preclinical assays.

Reproducible insulin secretion from isolated rat pancreas preparations using an organ bath.

Diabetes mellitus is a lifestyle-related disease that is characterized by inappropriate or diminished insulin secretion. Ex vivo pharmacological studies of hypoglycemic agents are often conducted using perfused pancreatic preparations. Pancreas preparations for organ bath experiments do not require cannulation and are therefore less complex than isolated perfused pancreas preparations. However, previous research has generated almost no data on insulin secretion from pancreas preparations using organ bath preparations. The purpose of this study was to investigate the applicability of isolated rat pancreas preparations using the organ bath technique in the quantitative analysis of insulin secretion from ß-cells. We found that insulin secretion significantly declined during incubation in the organ bath, whereas it was maintained in the presence of 1 µM GLP-1. Conversely, amylase secretion exhibited a modest increase during incubation and was not altered in the presence of GLP-1. These results demonstrate that the pancreatic organ bath preparation is a sensitive and reproducible method for the ex vivo assessment of the pharmacological properties of hypoglycemic agents.

In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles: Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency.

BACKGROUND: Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. METHODS: pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at -30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. RESULTS: Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. CONCLUSIONS: The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration.

Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia.

BACKGROUND AND AIMS: Heparin/protamine micro/nanoparticles (LH/P-MPs) were recently developed as low-molecular weight, biodegradable carriers for adipose-derived stromal cells (ADSCs). These particles can be used for a locally delivered stem cell therapy that promotes angiogenesis. LH/P-MPs bind to the cell surface of ADSCs and promote cell-to-cell interaction and aggregation of ADSCs. Cultured ADSC/LH/P-MP aggregates remain viable. Here, we examined the ability of these aggregates to rescue limb loss in a mouse model of hindlimb ischemia. METHODS: Unilateral hindlimb ischemia was induced in adult male BALB/c mice by ligation of the iliac artery and hindlimb vein. For allotransplantation of ADSCs from the same inbred strain, we injected ADSC alone or ADSC/LH/P-MP aggregates or control medium (sham-treated) directly into the ischemic muscles. Ischemic limb blood perfusion, vessel density, and vessel area were recorded. The extent of ischemic limb necrosis or limb loss was assessed on postoperative days 2, 7, and 14. RESULTS: Compared with the sham-treatment control, treatment with ADSCs alone showed modest effects on blood perfusion recovery and increased the number of α-SMA-positive vessels. Response to ADSC/LH/P-MP aggregates was significantly greater than ADSCs alone for every endpoint. ADSC/LH/P-MP aggregates more effectively prevented the loss of ischemic hindlimbs than ADSCs alone or the sham-treatment. CONCLUSION: The LH/P-MPs augmented the effects of ADSCs on angiogenesis and reversal of limb ischemia. Use of ADSC/LH/P-MP aggregates offers a novel and convenient treatment method and potentially represents a promising new therapeutic approach to inducing angiogenesis in ischemic diseases.

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.

Effective wound healing in streptozotocin-induced diabetic rats by adipose-derived stromal cell transplantation in plasma-gel containing fragmin/protamine microparticles.

We investigated the effectiveness of the application of inbred adipose-derived stromal cells (IR-ASCs) in high inbred rat plasma (IRP) (6%)-Dulbecco modified Eagle medium (DMEM) gel with fragmin/protamine microparticles (F/P MPs) (IR-ASCs + IRP-DMEM gel + F/P MPs) on wound healing in streptozotocin-induced diabetic rats. F/P MPs have previously been used as a cell carrier for IR-ASCs in inbred Fisher 344 rats and for preservation and controlled release of various cytokines in IRP-DMEM gel. We applied IR-ASCs + IRP-DMEM gel + F/P MPs to full-thickness skin excisions on the backs of the diabetic rats. The statistical significance of wound closure was evaluated on postwounding days 3, 7, 10, and 14, and the skin area surrounding the wound was removed for histological examination on days 7 and 14. The wound closure rate and histological examination of wounds treated with IR-ASCs + IRP-DMEM gel + F/P MPs demonstrated significantly advanced epithelialization, capillary formation, and granulation tissue formation. When DiI-labeled IR-ASCs + IRP-DMEM gel + F/P MPs were applied to full-thickness skin wounds on the backs of the diabetic rats, histological observation at 2 weeks showed appearances of both DiI-labeled granulation tissue and CD31-immunostained microvessels in the transplant areas. A portion of the transplanted IR-ASCs + IRP-DMEM gel + F/P MPs had been taken up into the granulation tissues to promote wound healing. Thus, IR-ASCs + IRP-DMEM gel + F/P MPs were effective for repairing healing-impaired wounds such as those arising in the diabetic rats.

Three-dimensional culture using human plasma-medium gel with fragmin/protamine microparticles for proliferation of various human cells.

Fragmin/protamine microparticles (F/P MPs) have been used as carriers for the preservation and activation of cytokines in human plasma (HP)-Dulbecco's modified Eagle's medium (DMEM) gels. This study investigated a three-dimensional (3D) culture system using an HP-DMEM gel with 0.1 mg/mL F/P MPs and 5 ng/mL FGF-2 for the proliferation of human dermal fibroblast cells (DFCs), human microvascular endothelial cells (MVECs) and human coronary smooth muscle cells (SMCs), or 5 ng/mL interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor (GM-CSF) for a human hematopoietic cell line (TF-1 cells). DFCs, MVECs, SMCs and TF-1 cells grew rapidly under 3D culture conditions using a low-concentration HP (2 %)-DMEM gel with F/P MPs and FGF-2 (for DFCs, MVECs and SMCs) or IL-3/GM-CSF (for TF-1 cells) at doubling times of 22, 23, 25 and 18 h, respectively, without the use of animal serum, compared to under 2D culture conditions using low-concentration human serum (2 %)-DMEM with 5 ng/mL FGF-2 or IL-3/GM-CSF on F/P MP-coated plates at doubling times of approximately 26, 25, 40 and 20 h, respectively.

Preparation of size-controlled silver nanoparticles and chitosan-based composites and their anti-microbial activities.

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.

Distribution of antimicrobial-resistant lactic acid bacteria in natural cheese in Japan.

To determine and compare the extent of contamination caused by antimicrobial-resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P=0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm-made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm-made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to >512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial-resistant LAB in imported and Japanese farm-made cheeses on the Japanese market, but not in Japanese commercial cheeses.

In vivo gene transfer using pDNA/chitosan/chondroitin sulfate ternary complexes: influence of chondroitin sulfate on the stability of freeze-dried complexes and transgene expression in vivo.

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.

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.