Takuya Horio

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

Water-insoluble fragmin/protamine microparticles of about 0.5-1 mum in diameter were prepared by simple mixing of low-molecular-weight heparin (fragmin) with protamine. We investigated the capability of these microparticles to immobilize fibroblast growth factor (FGF)-2, to protect FGF-2 against degradation, to enhance FGF-2 activity, and to facilitate controlled release of FGF-2. FGF-2 bound to the fragmin/protamine microparticles with high affinity (Kd = 2.08 x 10(-9) M) and the half-life of FGF-2-activity was prolonged substantially through binding of FGF-2 to the microparticles, by protection of FGF-2 from inactivation by heat and proteolysis. After subcutaneous injection into the back of mice, the fragmin/protamine microparticles underwent biodegradation and disappeared in about 2 weeks. A similar injection of FGF-2-containing microparticles resulted in significant neovascularization and fibrous tissue formation near the injection site after 1 week. These results indicate that controlled release of biologically active FGF-2 occurs through both slow diffusion and biodegradation of the microparticles, with subsequent induction of neovascularization. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009.

Enteral immuno‐enhanced diets with arginine are safe and beneficial for patients early after esophageal cancer surgery

We previously reported that provision of immediate enteral nutrition (EN) with a certain amount of omega (omega)-3 fatty acids (FAs) in patients after esophageal cancer surgery resulted in reduced platelet aggregation, coagulation activity, and cytokine production. We investigated whether EN using immuno-enhanced diet (IED) containing a large amount of omega-3 FAs as well as arginine and RNA affected the above-described responses. We also attempted to reveal whether arginine in the IED can potentially harm patients who undergo esophageal cancer surgery. Twenty-nine patients with esophageal cancer who underwent similar surgical procedures were selected. All patients received EN starting immediately after surgery. Fourteen patients received the formula with fewer omega-3 FAs, and fifteen patients received the IED. Administration of the IED tended to inhibit postoperative decrease in platelet count. Prothrombin activity and thrombin-antithrombin III complex levels were significantly reduced in the IED group. Plasma IL-8 levels were significantly lower (P < 0.05) in patients without the IED on the fifth postoperative day (POD). The proportion of T-cells was significantly higher (P < 0.05) in the IED group on PODs 1 and 7. Nitrate/nitrite levels did not differ significantly between the two groups. Early EN with an IED may enhance the inhibitory effects on postoperative platelet aggregation and hypercoagulation, and appeared to be advantageous to T-cell proliferation. These effects are expected to be beneficial in patients at risk of developing infectious complications. This study also showed that the IED could be safely used without any adverse effects for patients early after a radical surgery for the esophageal cancer.

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.

Duodenal gangliocytic paraganglioma with regional lymph node metastasis and a glandular component.

Gangliocytic paraganglioma (GP) is generally considered to be a benign periampullary lesion, although it is unclear whether it should be classified as a hamartoma or as a neoplasm. Here, we present a GP case with lymph node metastasis. A 16‐year‐old boy complained of exertional dyspnea. Upper endoscopy and imaging studies revealed a polypoid ampullary tumor. Pancreaticoduodenectomy with lymph node dissection was performed due to swelling of peripancreatic lymph nodes. Histologically, the tumor consisted of three cell types: epithelioid; spindle; and ganglion cells. In addition to these typical components of GP, a distinct glandular component was also present. There was substantial invasion of tumor cells into the lymphovascular vessels, associated with lymph node metastases. These lymph node metastases were histologically similar to the primary tumor. To judge from these findings GP may be a true neoplasm with metastatic capacity. Pre‐ and intraoperative investigations for lymph node or distant metastases are required for adequate resection of this kind of tumor.

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.

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.

Effects of platelet‐rich plasma‐containing fragmin/protamine microparticles in enhancing endothelial and smooth muscle cell growth and inducing collateral vessels in a rabbit model of hindlimb ischemia

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.

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.

Surgical attempts to avoid anastomotic leaks and reduce reflux esophagitis following esophagectomy for cancer

BackgroundWe analyzed the results of our surgical attempts to establish a safe reconstruction after esophagectomy for cancer that withstands both early and subsequent complications.MethodsPatients who underwent an intrathoracic or cervical esophagogastrostomy were selected. We preserved the esophagus keeping an oral margin of at least 3 cm and made an anastomosis with the gastric wall as low as possible to avoid an anastomotic leak. We included an antireflux procedure in the intrathoracic anastomosis. We examined the effect of these surgical approaches in three patient groups: one group with cervical anastomosis (CA group, n = 21), and the other two groups with intrathoracic anastomosis after resection of cancer in the upper or middle thoracic esophagus (UM group, n = 104) or in the lower thoracic or abdominal esophagus (LA group, n = 30).ResultsNo leak was found in the esophagogastric anastomosis in any group. A gastric suture line dehiscence developed in two cases in the UM group. Postoperative endoscopy revealed that mean anastomotic height in the UM group was 4.1 cm lower than in the CA group (P < 0.0001) and 2.1 cm higher than in the LA group (P = 0.0006). The incidence of reflux esophagitis was 0% in the CA group, 43% in the UM group, and 37% in the LA group, with significant differences between the CA group and the other groups.ConclusionsOur surgical attempts to avoid leaks of esophagogastrostomy were entirely successful. An intrathoracic anastomosis combined with an antireflux procedure was not advantageous for the incidence of reflux esophagitis compared to cervical anastomosis, but it minimized the effects of anastomotic height on the development of reflux esophagitis.