Sunao Kubota

Thermoreversible gelation on cooling and on heating of an aqueous gelatin–poly(N‐isopropylacrylamide) conjugate

A polymer conjugate composed of 43 wt% gelatin and 57 wt% poly(N-isopropylacrylamide) (PNIPAAm) was prepared. The dynamic viscoelastic properties of an aqueous solution of the conjugate at the concentration of 5 wt% were examined. The solution was viscous fluid at 30°C and turned into an elastic homogeneous hydrogel upon heating above 34°C or upon cooling below 10°C. The resultant hydrogels turned back into a solution at the opposite temperature cycles of the gelation. It is considered that the driving force of the gelation is the intermolecular hydrophobic interaction of PNIPAAm blocks or the intermolecular helix association of gelatin blocks, respectively, on heating or on cooling.

Selective cell death by water-soluble Fe-porphyrins with superoxide dismutase (SOD) activity

We investigated the effect on cell death of reactive oxygen species induced by [[5,10 (or 5,15)-bis(N-methyl-4-pyridyl)-15,20 (or 10,20) diphenyl]porphinato]iron (cis-FeMPy(2)P(2)P or trans-FeMPy(2)P(2)P) with SOD activity. The SOD activities of the cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P were measured using stopped-flow kinetic analysis. The cell viability of four cell lines treated with cis-Fe-porphyrin, trans-Fe-porphyrin, mitomycin c (MMC), or cisplatin was estimated by the alamar blue exclusion assay of the modified MTT method. The amount of cis-FeMPy(2)P(2)P and trans-FeMPy(2)P(2)P in the Walker 256 cultured for 24 h was 4.0 and 2.6 fmolcell(-1), respectively, indicating that the plasma membrane permeability of the Fe-porphyrins depended on their structure. Cis-FeMPy(2)P(2)P selectively killed Walker 256 and H-4-II-E as cancer cells but not FR and BRL-3A as normal cells and showed a significant cytotoxicity for the cancer cells compared with trans-FeMPy(2)P(2)P, MMC and cisplatin. We believe that cis-FeMPy(2)P(2)P as an SOD mimic converts intracellular O(2)(*-) to H(2)O(2) and that H(2)O(2) or *OH causes DNA damage and induces cell death. This result suggests that for the SOD mimic, O(2)(*-) may be useful as a target molecule to induce selective cell death between cancer and normal cells and that a metalloporphyrin having SOD activity is a new class of anticancer agents.

Evaluation of Thermoreversible Gelation Polymer for Regeneration of Focal Liver Injury

Liver injuries are often associated with complications including infection of the dead space, bleeding, leakage of bile and so on. We have recently developed a thermoreversible gelation polymer (TGP) which provides a good healing environment for wounds and possibly reduces complications. The purpose of this study was to evaluate whether adequate regeneration occurred with a liver defect by using TGP. The sol-gel transition of TGP is reversibly controlled by temperature; TGP is soluble below a lower critical solution temperature (LCST) of 22°C, and becomes solid above the LCST. Soluble TGP can reach anywhere, and gelation of TGP occurs at the wound surface by body temperature to fill the wound/cavity. A section of median part of the left lobe comprising 3% (2 × 2 cm wide and 1 cm deep) of the liver was resected, and the Beagle dogs were assigned to three groups: ‘resection alone group’, ‘resection + fibrin glue (FG) group’ and ‘resection + TGP group’. The resection alone group and the resection + FG group showed severe fibrosis at week 12, and a scarring was clearly visible. The resection + TGP group showed almost complete healing by week 4, with no adhesion and recession of the wound; the resection site was completely filled with TGP, liver-like capsule organoids emerged to cover the wound and neovascularization was observed within the organoids. Furthermore, the resected liver regenerated completely by week 12, TGP was replaced by hepatocytes, and the presence of hepatic lobules confirmed structural reorganization. The number of RCA-1-positive macrophages accumulating around the wound was significantly reduced in the resection + TGP group compared to the other two groups. In the early stage of liver resection and regeneration, TGP seemed to suppress the accumulation of macrophages and stellate cells. In the late stage, when massive inflammatory cell accumulation had subsided, TGP was degraded, that may contribute to avoid unnecessary inhibition of the liver regeneration process. Collectively, TGP may induce efficient regeneration by reducing the fibrosis and enhancing proliferation, even with a minor liver defect. Because TGP has good biocompatibility, it may become useful as an ideal biomaterial for the treatment of liver injuries.

Thermoreversible gelation polymer induces the emergence of hepatic stem cells in the partially injured rat liver.

Focal injury of the adult liver causes formation of granulomatous tissue and fibrosis. When thermoreversible gelation polymer (TGP) was applied to such defects of the rat liver, complete recovery of hepatic tissues was observed without granulation. We analyzed the mechanism of the regeneration. TGP is a chemically synthesized biocompatible polymer material whose sol‐gel transition is reversible by changing the temperature. Cooled TGP was poured into a penetration lesion of the rat liver. Immunohistochemistry and polymerase chain reaction were carried out using tissues and cultured cells isolated from ductular structures. Immunocytochemical and ultrastructural analyses were also conducted. Seven days after TGP treatment, ductular reactions were observed around the wound and ductules elongated to the injured area. Cells in the structures were alpha‐fetoprotein (AFP) positive, albumin+, CK19+, c‐Kit+, and Thy1+. Hepatocyte‐like cells possessing glycogen appeared around the tips of the ductules from day 9. The defect was completely replaced with hepatocytes by day 28. Cells isolated from the ductules expressed Musashi‐1, c‐Kit, Thy1, AFP, albumin, transferrin, connexin 43, and CK19. When the cultured cells were covered by TGP, they rapidly proliferated to form colonies, whereas without TGP cells gradually died. Morphologically and ultrastructurally the cells were similar to hepatocytes. They expressed not only albumin and transferrin but TAT, CYP2E1, and CCAAT/enhancer binding protein α. Some cells formed bile canaliculus‐like structures. In conclusion, TGP may trigger the initiation of hepatic stem cells in biliary ductules, and stem cell activation may occur even in the regeneration of the normal liver. (HEPATOLOGY 2006;43:1053–1062.)

Development of a novel polyimide hollow fiber for an intravascular oxygenator

The authors have synthesized a novel fluorinated polyimide to develop a membrane material for oxygenators and fabricated polyimide hollow fibers for use in an intravascular oxygenator. A dry/wet phase inversion process has been applied to a spinning process to prepare an asymmetric polyimide hollow fiber. The outer surface of the hollow fiber consists of an ultrathin, dense skin layer, with a calculated apparent thickness of ∼60 μ. The fiber diameter was 800 μ with a wall thickness of 130 μ. The asymmetric hollow fiber has two advantages because (a) the hollow fiber does not produce plasma leakage due to the dense skin layer of the surface and (b) O2 and CO2 transfer rates through the hollow fiber are enhanced due to the ultrathin skin layer and are significantly larger than those of presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber without heparinization has been evaluated in vitro. Deformation and aggregation of platelets adherent to the fibers were not observed, and the polyimide suppressed platelet activation. The polyimide significantly reduced the production of anaphylatoxin and also suppressed complement activation. ASAIO Journal 1997; 43:M490-M494.

Evaluation of blood compatibility of fluorinated polyimide by immunolabeling assay

Aromatic fluorinated polyimide (6FDA-6FAP) derived from 2,2′-bis(3,4-dicarboxyphenyl) hexafluoropropane dianhydride (6FDA) and 2,2′-bis(4-aminophenyl) hexafluoropropane (6FAP) have been synthesized with chemical imidation for a novel membrane oxygenator. In this study we investigated the interactions of the fluorinated polyimide with plasma protein, platelets, neutrophils, and complement, which influence thrombus formation and the activation of the immune system. The immunogold labeling technique was used for the detection of adsorbed proteins. One of the key findings is that the adsorption of albumin (Alb) and fibrinogen (Fib) from plasma on the surface of the heat-treated polyimide was significantly suppressed. However, the adsorption of γ-globulin (IgG) with an isoelectric point of approximately pH 7 onto the polyimide surface was selectively facilitated. The orientation of the adsorbed IgG was determined by using anti-human IgG (Fab′)2, and it was determined that almost all the IgG was adsorbed on the surface of the polyimide through the Fc fragment by hydrophobic interaction. These findings will be partially associated with the strong surface negativity combined with hydrophobicity of the polyimide surface, and elucidate the results of the suppressed platelet adhesion and Fc-mediated immune activation of the polyimide.

Ductular Cell Proliferation in Islet Cell Neogenesis Induced by Incomplete Ligation of the Pancreatic Duct in Dogs

PurposeIt has been suggested that islet neogenesis can be induced by incomplete ligation of the pancreatic duct in small animals; however, there has been no report of neogenesis and the proliferation of islets occurring in larger animals. When this procedure was performed in the Vervet monkey, it produced a noticeable increase in duct proliferation, but islet neogenesis was not observed, although the number of monkeys examined was very small. We conducted this study to evaluate whether islet neogenesis and ductular proliferation could be induced in larger animals such as the dog, by partial obstruction of the pancreatic duct.MethodsIncomplete ligation of the pancreatic duct was induced by tying the pancreas around the ventral side of the head with 2-0 silk and reducing the circumference by about 80% to cause partial obstruction.ResultsBy 2 weeks after ligation, we saw hyperplasia of the epithelial cells, multilayering of cuboidal cells, and proliferation of ductular cells. The terminal ductules involved in the formation of immunohistochemically insulin-positive islets, and islets, formed adjacent to the alignment of the ductular cells. By 8 weeks after ligation we saw scattered islets, less than 50 µm in diameter and less than 1 000 µm2 in area. These cells were immunolabeled for both insulin and cytokeratin, and there was continuity between these insulin-positive cells and terminal ductular cells. Glucagon-positive cells and somatostatin-positive cells were also found adjacent to the alignment of ductular cells.ConclusionsThese results suggest that islets may be differentiated from precursor cells in the pancreatic duct, and that stem cells exist even in adults.

Development of a fluorinated polyimide hollow fiber for medical devices.

Abstract We fabricated an asymmetric polyimide hollow fiber for medical devices. A dry/wet phase inversion process was applied to a spinning process to prepare the hollow fiber. The outer diameter was 330 μm with a wall thickness of 70 μm. Transfer rates of O2 and CO2 in the asymmetric polyimide fiber were 6.9 × 10−3 and 5.5 × 10−3 cm3 (STP)/(cm2 s cmHg), respectively, which are approximately 10 times higher than those measured in the Menox and Si-polypropylene fibers of the presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber was evaluated in vivo, indicating that polyimide had excellent blood compatibility when compared with silicone-coated fiber. Additionally, we fabricated a novel porous membrane with three-dimensional fine structure from cylindrical microscale pores and examined possibility of a porous membrane for use in hemodialysis.

Gas transfer and blood compatibility of asymmetric polyimide hollow fiber

We have fabricated an asymmetric polyimide hollow fiber for use as a membrane oxygenator. A dry/wet phase inversion process has been applied to a spinning process to prepare the hollow fiber. The fiber structure consisted of a complete defect-free skin layer and a porous substructure characterized by the presence of an open-cell structure and macrovoids. The outer diameter was 480 μm with a wall thickness of 50 μm. Transfer rates of O2 and CO2 in the asymmetric polyimide fiber were 2.3 × 10-5 and 1.1 × 10-4 (cm3(STP)/(cm2s cmHg)), respectively, which were four times higher than those measured in the polydimethylsiloxane (PDMS) fiber of the presentlyavailable membrane oxygenator. The (QO2 /QN2)selectivity of the polyimide fiber was 4.9, indicating that the surface skin layer is essentially defect-free. The blood compatibility of the polyimide hollow fiber has been evaluated in vitro and in vivo. The polyimide had an excellent blood compatibility when compared with PDMS.

A new chemosensitivity assay for ascites tumor cells using a thermoreversible gelation polymer as a culture medium and the observed clinical responses.

Background/Aim: Ascites tumor cells from patients with peritonitis carcinomatosa were tested for cis-diamminedichloroplatinum (CDDP) sensitivity. The patients were divided into CDDP-sensitive and resistant groups. Survival and time to progression (TTP) rates were compared. Materials and Methods: 18 peritonitis carcinomatosa patients with class V ascites based on cytologic diagnosis were enrolled in this study. Chemosensitivity testing of the ascites tumor cells was done to determine their sensitivity to CDDP using a three-dimensional culture matrix of thermoreversible gelation polymer (TGP). CDDP at a dose calculated to achieve ascitic fluid drug levels equivalent to the IC50 was given intraperitoneally to 12 CDDP-sensitive patients and 6 CDDP-resistant patients. Results: Both the median survival time and the median TTP were significantly longer in CDDP-sensitive patients than in CDDP-resistant patients (survival time 105 vs. 13 days, p = 0.019; TTP 90 vs. 5 days, p = 0.029). Conclusion: The results indicate the potential feasibility of controlling ascites in cancer patients in whom a maximal dose effect can be achieved with a minimal dose of chemotherapy.