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.

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.

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.

CDDP-induced desmoplasia-like changes in oral cancer tissues are related to SASP-related factors induced by the senescence of cancer cells.

The tumor microenvironment (TME) concept has been proposed and is currently being actively studied. The development of extracellular matrix (ECM) in the TME is known as desmoplasia and is observed in many solid tumors. It has also been strongly associated with poor prognosis and resistance to drug therapy. Recently, cellular senescence has gained attention as an effect of drug therapy on cancer cells. Cellular senescence is a phenomenon wherein proliferating cells become resistant to growth-promoting stimuli, secrete the SASP (senescence-associated phenotypic) factors, and stably arrest the cell cycle. These proteins are rich in pro-inflammatory factors, such as interleukin (IL)-6, IL-8, C-X-C motif chemokine ligand 1, C-C motif chemokine ligand (CCL)2, CCL5, and matrix metalloproteinase 3. This study aimed to investigate the desmoplasia-like changes in the TME before and after cancer drug therapy in oral squamous cell carcinomas, evaluate the effect of anticancer drugs on the TME, and the potential involvement of cancer cell senescence. Using a syngeneic oral cancer transplant mouse model, we confirmed that cis-diamminedichloroplatinum (II) (CDDP) administration caused desmoplasia-like changes in cancer tissues. Furthermore, CDDP treatment-induced senescence in tumor-bearing mouse tumor tissues and cultured cancer cells. These results suggest CDDP administration-induced desmoplasia-like structural changes in the TME are related to cellular senescence. Our findings suggest that the administration of anticancer drugs alters the TME of oral cancer cells. Additionally, oral cancer cells undergo senescence, which may influence the TME through the production of SASP factors.

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.

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.

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

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

Delivery system for autologous growth factors fabricated with low-molecular-weight heparin and protamine to attenuate ischemic hind-limb loss in a mouse model.

Frozen and thawed platelet-rich plasma (PRP) contains high concentrations of various growth factors, such as fibroblast growth factor (FGF)-2, vascular endothelial growth factor, and hepatocyte growth factor. We previously reported that low-molecular-weight heparin/protamine microparticles (LH/P MPs) are useful as biodegradable carriers for the controlled release of FGF-2. In this study, we examined the ability of PRP/LH/P MPs to prevent limb loss in an induced ischemic hind-limb model that used adult BALB/c-nu/nu male mice. One day after inducing ischemia, intramuscular injections of a PRP/LH/P MPs solution were administered into several sites of the ischemic hind limb. Seven days and onward after the injections, the PRP/LH/P MPs-treated and PRP-treated groups recovered from ischemia, as reflected by the improved oxygen saturation. In the PRP-treated group, however, the level of recovery of oxygen saturation after ischemia decreased after 14 days. From the 21st day onward, there was a significant difference between those two groups. In the LH/P MPs-treated group, a partial recovery occurred only in the early period. The saline-treated group (i.e., the control) and the noninjection group (i.e., ischemia only) exhibited no recovery. The limb survival rate at 1 year in the ischemia-induced mice injected with PRP/LH/P MPs was approximately 25 % (two of eight mice) but was absent in the other groups.

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.

Efficacy of fragmin/protamine microparticles containing fibroblast growth factor-2 (F/P MPs/FGF-2) to induce collateral vessels in a rabbit model of hindlimb ischemia.

OBJECTIVES: The 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. METHODS: A 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. RESULTS: Neither 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. CONCLUSIONS: The 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.

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.

Enhanced effect of platelet-rich plasma containing a new carrier on hair growth.

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

Enhanced healing of mitomycin C-treated 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.

Effect of photocrosslinkable chitosan hydrogel and its sponges to stop bleeding in a rat liver injury model.

This study examined the hemostatic efficacy of photocrosslinkable chitosan hydrogel-mixed photocrosslinked chitosan sponges (PCM-S) after hepatic injury in rats. The left lobe of the liver was penetrated with a dermal punch to produce a penetrating wound in heparinized and nonheparinized rats. Treated rats either had PCM-S applied into the wound and then were immediately ultraviolet irradiated, or they had TachoComb (TC) inserted into the wound. Blood loss, hemostasis, and survival were quantified after the hepatic injury. Measurements on serum alanine aminotransferase in nonheparinized rats and hemoglobin concentrations and histologic examinations in heparinized rats were performed to assess hepatic function. Although the hemostatic effect in the PCM-S-treated nonheparinized rats was identical to that of the TC-treated group, PCM-S-treatment has higher hemostatic effect in heparinized rats. No adverse events related to the use of PCM-S were detected in blood and histologic examinations.

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.

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.

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

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

Fragmin/protamine microparticle-coated matrix immobilized cytokines to stimulate various cell proliferations with low serum media.

Fragmin/protamine microparticles (F/P MPs) have been shown to bind to culture plates, thereby retaining heparin-binding cytokines. Most protocols for in vitro cultures of human microvascular endothelial cells (hMVECs), human dermal fibroblast cells (hDFCs), and hematopoietic cell line (TF-1) include high fetal bovine serum (FBS) (10%) medium as a nutritional supplement. Growth rates of those cells on the F/P MP-coated plates were higher in low FBS (1%) medium containing fibroblast growth factor (FGF)-2 (for hMVECs and hDFCs) and interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor (for TF-1 cells) than without coating. The cytokines in low FBS medium were shown to be immobilized on the F/P MP-coated plate and released into the culture medium with a half releasing time of 4-5 days. Furthermore, those cells grew well on each cytokine-preimmobilized F/P MP-coated plate in low FBS medium. Thus, the F/P MP-coated matrix with adequate heparin-binding cytokines may provide biomaterials for controlling cellular growth and differentiation.