Makoto Kodama

Pore size, tissue ingrowth, and endothelialization of small-diameter microporous polyurethane vascular prostheses

Small-diameter microporous polyurethane vascular prostheses with an average pore size of between 5 and 30mum at the outer surfaces and 30mum at the luminal surface were prepared. Thirty-two PU and 8 expanded polytetrafluoroethylene (ePTFE) prostheses were implanted into the abdominal aorta of rats for periods ranging from 1 to 8 weeks. Harvested prostheses were analysed histologically and morphologically. The progress of endothelial-like cells and the extent of infiltration of perigraft tissues were quantified. All of the prostheses showed fast growth of endothelial-like cells in the second week, with the PU prostheses having an external pore size of 30mum producing the highest rate. It was also during the second week that perigraft tissue grew most significantly into the prosthetic structure. This coincident may suggest the importance of rapid tissue regeneration for the early endothelial healing. The role of the ingrowth perigraft tissues is likely to support and stabilize the neointima. The thickening of neointima was mainly located at the vicinity of the proximal anastomoses of some of the PU prostheses and was unrelated with the extent of perigraft tissue infiltration. In the PU prostheses, a complete lining of endothelial-like cells was achieved by the end of 4 weeks. Expanded PTFE prostheses displayed smooth, thin intima, very limited tissue ingrowth, and incomplete coverage of endothelial-like cells.

Polyacrylamide containing sugar residues : synthesis, characterization and cell compatibility studies

Hydrophilic poly (acrylamide) compounds having simple mono saccharides (such as glucose and galactose) as pendent groups were synthesized. Cell culture polystyrene plates were coated with these polymers. A distinct transition for water contact angle from higher to a lower value was noted for coated plates. FTIR-ATR of coated plates showed a characteristic band at 1653 cm-1, 1706 cm-1 and 1611 cm-1 due to amide carbonyl for native, glucose and galactose poly (acrylamides) respectively. The XPS spectra of cell culture plates coated with native and sugar derivatives of poly (acrylamides) showed peaks around 277, 287 eV(C Is), 401 eV(N Is) and 525, 534 eV(O Is). The growth rate of mouse fibroblast L929 cells was found to be higher on poly (acrylamide) having galactose residues than that of glucose. A high degree of cell aggregation was also observed in case of galactose poly (acrylamides).

Cryopreservation of primarily isolated porcine hepatocytes with UW solution.

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbeccos modified Eagles medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

Anticoagulant surface prepared by the heparinization of ionic polyurethane film

We present a new method for heparinization on the surface of polyurethane. The segmented polyurethane was first modified with an epoxide monomer and followed by a ring-opening reaction with diethanolamine to introduce sufficient hydroxyl groups on the surface of cast film. On this film surface, a cationic monomer was grafted by using tetravalent Cerium salt as an initiator. Heparin was immobilized in high efficiency on the ionized surface through static interactions in aqueous solution. The structure of ionized and heparinized surfaces were characterized by attenuated total reflectance infrared spectroscopy (ATR–FTIR) and electron spectroscopy for chemical analysis (ESCA) spectra. The platelet-rich plasma (PRP) contacting test and the platelet-poor plasma (PPP) clotting time measurements showed that the immobilized heparin retained its strong anticoagulant property. The release of heparin from film into salt solution was also studied, and it was found that only a small portion of heparin (10–20%) was released over a period as long as 10 h. It is expected that this new method for surface heparinization can be used to prepare antithrombogenic materials with long-term stability.

Hepatocyte isolation and transplantation in the pig.

Hepatocyte transplantation (HTX) has received great expectation for the treatment of a wide spectrum of liver diseases. Considering the severe shortage of human livers for hepatocyte isolation, porcine hepatocytes are an attractive alternative to normal human hepatocytes. To develop such therapy, establishment of an efficient hepatocyte isolation and transplantation model that enables accurate assessment of safety and efficacy of HTX is extremely important. Porcine hepatocytes were isolated from a surgically removed liver segment with a four-step retrograde perfusion using dispase and collagenase. The resultant hepatocytes of > 84% viability were used for transplantation experiment in a pig model of acute liver failure induced by intravenous administration of D-galactosamine (D-gal) (0.5 mg/kg). Twenty-four hours after D-gal injection, transplantation of freshly isolated porcine hepatocytes (1 x 10(9)) was safely conducted and prolonged the survival of D-gal-treated pigs. We describe an efficient porcine hepatocyte isolation and subsequent cell transplantation in pigs with D-gal-induced liver failure.

Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside.

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 μg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbeccos modified Eagles medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 μg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 μg/ml) would be a useful cold preservation means for the development of cell therapies.

Study of blood compatible polymers: III. Copolymers of N-benzyl, N-(2-hydroxyethyl) acrylamide and 2-hydroxyethyl methacrylate

Copolymerization of 2-hydroxyethyl methacrylate (HEMA) and N-benzyl, N-(2-hydroxyethyl) acrylamide (BENAAm) was carried out at different mole ratios of the monomers to obtain copolymers of varying composition. BENAAm content of the copolymers varies between 13 and 70%. Investigation of the interaction of rabbit platelets with these polymer surfaces showed that copolymers with higher BENAAm content inhibit the platelet deformation. Human umbilical cord fibroblast cells proliferated very well on the copolymer surfaces. The cell growth rate on polyHEMA was relatively low. Maximum cell growth was observed on the copolymer having 87% HEMA.

Platelet adhesion and complement activation studies on poly(N-alkyl mono and disubstituted) acrylamide derivatives

Poly(N-alkyl mono and disubstituted) acrylamide derivatives were synthesized from poly(acryloyl chloride) by monomer analogous reaction. The polymers were characterized by FTIR-ATR and GPC. The contact angle measurements were performed to evaluate hydrophobic/hydrophilic characters of these polymers. The N-alkyl substituents changed contact angle between 55 and 75 degrees. In vitro platelet adhesion studies showed that surfaces of poly(N-alkyl substituted) acrylamides are prone to adhere platelets. Platelet spreading was more on poly(N-benzyl-N-ethyl acrylamide) surfaces in comparison to that on poly(N-benzyl-N-propionic acid ethyl ester acrylamide) and poly(N-benzyl acrylamide) surfaces. As a result of modification of amino group with N-alkyl substituents, the activations of C3a and C5a complements were suppressed 9-20% and 5-6% of native poly(acrylamides), respectively.

Synthesis, characterization and blood compatibilities of novel segmented polyurethanes containing poly(butadiene) soft segments and phosphatidylcholine analogues in the main chains and long-chain alkyl groups in the side chains

New segmented polyurethanes (SPUs) containing poly(butadiene) (PBD) soft segments and phospholipid moieties in the main chains and long-chain alkyl groups in the side chains were synthesized. The phospholipid moieties include bis[2-(2-hydroxyethyldimethylammonio)ethyl] 2-octylpropane-1,3-diyl bis(phosphate) (OcPDP), bis[2-(2-hydroxyethyldimethylammonio)ethyl] 2-oleylpropane-1,3-diyl bis(phosphate) (OlPDP) and bis[2-(2-hydroxyethyldimethylammonio)ethyl] 2-cetylpropane-1,3-diyl bis(phosphate) (CPDP). The bulk characterization of synthesized SPUs was investigated by infrared (IR) spectroscopy and gel-permeation chromatography (GPC). The mechanical properties were evaluated by dynamic viscoelasticity and tensile measurements. The existence of phospholipid analogous groups on the surface of these SPUs was revealed by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The blood compatibilities of the new polymers were evaluated by platelet rich plasma (PRP) contact studies and viewed by scanning electron microscopy (SEM) using medical grade BioSpan ® and non-phospholipid polyurethane as references. These new materials have good surfaces in terms of platelet adhesion, and the morphology of adhered platelets undergoes a relatively low degree of variation.

Polyacrylamides containing sugar residues : synthesis, characterization and hepatocyte attachment studies

Homo polymers of acrylamide having glucose (PAAm-glucose) and galactose (PAAm-galactose) as pendent groups were synthesized. Tissue culture polystyrene (TCPS) plates coated with these polymers showed increased surface wettability. Coating of PAAm-glucose and PAArn-galactose on to TCPS plates was also confirmed by X-ray photoelectron spectroscopic (XPS) characterization. Rat hepatocytes in primary culture attached to the surfaces of PAArn-galactose homopolymer, but not to those of PAAm-glucose homopolymer.