Xyloglucan as a synthetic extracellular matrix for hepatocyte attachment

The possibility of employing naturally derived xyloglucan (XG) having galactose moieties in the side chain for the development of synthetic extracellular matrix in tissue engineering was studied. Hepatocyte adhesion to the XG-coated polystyrene (PS) dish was 73.9% after 30 min incubation, whereas that to the PS dish as a negative control was 59.1%. The hepatocyte adhesion to the XG-coated surface was dependent on the presence of Ca2+ ions, whereas that to the XG-coated surface could not be induced by Mg2+ ions alone, indicating specific interaction between galactose moieties of XG and asialoglycoprotein receptors of hepatocytes. From the results of fluorescence, confocal laser micrographs and flow cytometry, it was suggested that XG was internalized by hepatocytes through a receptor-mediated mechanism. The DNA synthesis of hepatocytes attached to the XG-coated surface was decreased with an increase of the coating concentration of XG and in the presence of epidermal growth factor (EGF). The spreading shapes of the hepatocytes attached to the surface in the presence of EGF at low concentration of XG (1 microg/ml) were enhanced. The hepatocytes attached to the surface at a high concentration of XG (200 microg/ml) showed round shapes with spheroids after 16 h in the presence of EGF.     

Epidermal Growth Factor Behaves as a Partial Agonist in Hepatocytes: Effects on DNA Synthesis in Primary Culture and Competition with Transforming Growth Factor α

Abstract

The structurally related mitogens epidermal growth factor (EGF) and transforming growth factor a (TGFα) are believed to exert all their effects via the same receptor. We have compared the effects of EGF and TGFα, and examined their interaction, on DNA synthesis in cultured rat hepatocytes. The potency of the two agents was similar, or slightly higher for EGF, but TGFα stimulated the DNA synthesis more efficiently, producing at high levels a rate of S phase entry that clearly exceeded (two to threefold) that obtained with maximally effective concentrations of EGF. While the hepatocytes became more sensitive both to TGFα and EGF when addition of the agents was postponed until late in the prereplicative period, TGFα exhibited higher efficacy than EGF both at early and late exposure. When EGF and TGFα were added together at 24 h, TGFα further enhanced the DNA synthesis in the presence of a saturating concentration (5 nM) of EGF, while EGF dose-dependently reduced the DNA synthesis in the presence of a high concentration (10 nM) of TGFα. The results show a lower efficacy of EGF than of TGFα, and, therefore, EGF displays the characteristics of a partial agonist in its EGF receptor-mediated growth stimulation in hepatocytes.     

Alginate/galactosylated chitosan/heparin scaffold as a new synthetic extracellular matrix for hepatocytes

Formation of multicellular hepatocyte spheroids in the three-dimensional culture is a potential approach for enhancing liver-specific functions in bioartificial liver (BAL) devices. In this study, as a synthetic extracellular matrix (ECM) for hepatocytes, a highly porous hydrogel (sponge-like) scaffold, 150-200 microm pore size in diameter, was fabricated with alginate (AL), galactosylated chitosan (GC), and heparin through electrostatic interaction. We attempt to select the best condition of AL/GC/heparin sponges for coculture with NIH3T3, as well as compare the liver-specific functions with monoculture. Cell adhesion to GC based on AL film was significantly increased with increasing GC concentration, but not to chitosan regardless of its concentration. The optimal concentration of GC and heparin in AL/GC/heparin sponges to perform the best liver-specific function was 1 and 6 wt% to AL contents, respectively, where albumin secretion were maintained with maximal rates. The mechanical properties in tensile strength of three types of sponges were very slightly different from one another. Cell viabilities performed on AL, AL/GC, and AL/GC/heparin sponges were 68.5, 83.3, and 90.4 % of control, respectively, after 15 days of incubation. Hepatocyte spheroids were more rapidly formed in the AL/GC and AL/GC/heparin sponges, with diameter enlarged to about 100 microm, than in AL sponges. Connexin32 and E-cadherin genes correlated with cell-to-cell adhesion were expressed in hepatocytes within AL/GC and AL/GC/heparin sponges at 36 h after incubation, but not in AL sponges. Treatment of a gap junctional intercellular communication (GJIC) inhibitor, 18beta-glycyrrhetinic acid, indicates that cell aggregation without GJIC does not perform the liver-specific functions for long periods. In the presence of HGF, the level of albumin secretion in AL/GC/heparin sponges was markedly elevated compared to that in AL/GC sponges. Coculture of hepatocytes in AL/GC/heparin sponges with NIH3T3 in a transwell insert resulted in significant increase of liver-specific functions, such as improved albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1 compared to those in hepatocyte monoculture. The results suggest that hepatocytes as stable spheroids enhance liver-specific functions in AL/GC/heparin sponges, providing a new synthetic ECM to design BAL devices.     

Enhanced liver functions of hepatocytes cocultured with NIH 3T3 in the alginate/galactosylated chitosan scaffold

Formation of primary hepatocyte spheroids in the hydrogel scaffold is a promising approach for enhancing liver-specific functions in liver tissue engineering as well as for developing bioartificial liver (BAL) devices. In the present study, a highly porous hydrogel scaffold composed of alginate (AL) and galactosylated chitosan (GC) as a synthetic extracellular matrix (ECM) for hepatocytes was fabricated with 150-200 microm pore size in diameter. Cell adhesion onto AL/GC and AL/chitosan film was 72.7 and 45% at 1 wt% of GC (or chitosan) to AL content whereas cell adhesion onto AL film was 28.5%. The optimal concentration of GC in AL/GC sponge was 1 wt% to AL content by the measurement of albumin secretion. Cell viabilities performed on AL and AL/GC sponges were 72.2+/-3.6 and 81.3+/-3.5% of control, respectively, after 10 days incubation. Hepatocytes were aggregated to form multicellular spheroids in AL/GC sponge with diameter enlarged up to about 100 microm, 36 h postseeding, whereas most of them in the AL sponge remained as single cells and only a few cells began to form aggregates. Intercellular molecules such as connexin32 and E-cadherin genes related with cell-cell contact were expressed in hepatocytes within AL/GC sponge at 36 h after incubation, but not in AL sponge. Treatment with a gap junctional intercellular communication (GJIC) inhibitor, 18beta-glycyrrhetinic acid, resulted in a 1.5-fold marked decrease in albumin secretion levels in AL/GC sponge. Specially, coculture of hepatocytes in AL and AL/GC sponges with NIH3T3 in a transwell insert resulted in enhanced increase of liver-specific functions, such as albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1, compared to those in hepatocyte monoculture. The results suggest that formation of hepatocyte spheroids in coculture system enhances liver-specific functions for the AL/GC sponge as a new synthetic ECM to design developed BAL devices.     

Morphology and metabolism of Ba-alginate-encapsulated hepatocytes with galactosylated chitosan and poly(vinyl alcohol) as extracellular matrices

Lactobionic acid, bearing a beta-galactose group, was coupled with chitosan to provide synthetic extracellular matrices together with poly(vinyl alcohol) (PVA). The hepatocytes encapsulated in Ba-alginate capsules with galactosylated chitosan (GC) and PVA as extracellular matrices showed aggregation morphologies as the incubation time increased. Ba-alginate-encapsulated hepatocytes with GC exhibited a higher metabolic function in albumin secretion compared to those entrapped in Ba-alginate beads and monolayer-cultured on a collagen-immobilized polystyrene dish. The ammonia removal ability of monolayer-cultured hepatocytes decreased with increasing culture time and disappeared completely after three days. In contrast, the ammonia removal ability of encapsulated and entrapped hepatocytes increased with increasing incubation time in the first seven and five days, respectively. Thereafter, the entrapped hepatocytes lost ammonia removal ability quickly while the encapsulated hepatocytes kept a relatively high ammonia removal ability up to 13 days. The trace amount of GC in the core matrices enabled encapsulated cells to enhance their ammonia removal and albumin secretion ability. The results obtained with 3-(3,4-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) also showed that the capsules incorporated with GC can provide a better microenvironment for cell aggregation along with nutrition and metabolite transfer. Due to the nature of the liquid core, the encapsulated hepatocytes showed very good mobility. This facilitated cell-cell interaction and cell-matrix interaction.     

A short low-level exposure to metavanadate during a cell cycle-specific interval of time is sufficient to permanently derange the differentiative properties of Mel cells

Mouse erythroleukemia (Mel) cells have a cell cycle-dependent high sensitivity to chemical and physical mutagens. This report shows that a 5 h exposure to 0.1 or 0.01 microg/ml metavanadate during the initial period of erythroid differentiation induction was sufficient to permanently damage the ability of treated Mel cells and their progeny to undergo erythroid differentiation, without affecting cell viability and proliferation. Conversely, a 5 h pulse of metavanadate at 1 or 10 microg/ml inhibited both differentiation and cell proliferation. The cell cycle-dependent period of mutagenesis was essential for fixation of damage in the cell genome and the progeny of the cells treated with 0.1 or 0.01 microg/ml metavanadate stably inherited an impaired capacity to differentiate. The efficiency of the DNA repair synthesis machinery during the specific period of exposure of Mel cells seemed directly involved in damage fixation. In fact, the mutagenic effects of a 0.1 microg/ml metavanadate pulse was further increased in the presence of 1 mM hydroxyurea, an inhibitor of DNA repair synthesis. In contrast, 5 microg/ml vanillin, an antimutagenic agent that stimulates repair, completely restored the capacity of progeny of cells treated with 0.1 microg/ml metavanadate to complete differentiation. Determination of [(3)H]deoxythymidine in acid-insoluble DNA indicated that incorporation was stimulated by metavanadate alone and was further increased by metavanadate plus vanillin; conversely, incorporation of thymidine was reduced in the presence of hydroxyurea. The capacity of metavanadate to permanently damage Mel cell erythroid differentiation appeared to depend on the cell cycle-related efficiency of the DNA repair systems, activated to correct the induced alteration, rather than on a specific concentration.     

Galactosylated chitosan/DNA nanoparticles prepared using water-soluble chitosan as a gene carrier

Water-soluble chitosan (WSC) was used to increase the stability of chitosan in water and decrease the cytotoxicity induced by acetic acid. Lactobionic acid (LA) bearing galactose group was coupled with WSC for hepatocytes specificity. The composition of galactose in galactosylated chitosan (GC) was determined by NMR spectroscopy. The GC was complexed with plasmid DNA in various GC/DNA (N/P) charge ratios and the resulting complex was characterized by dynamic light scattering, gel retardation, and turbidity to determine the particle sizes, complex formation, and complex stability, respectively. Cytotoxicity and transfection efficiency of GC were also studied in cultured HepG2 human hepatoblastoma cell line and HeLa human cervix epithelial carcinoma cells. The complete GC/DNA complex was formed at the charge ratio of 5 and the GC/DNA complex to DNase I resistance was obtained. Particle sizes decreased with increasing charge ratio of GC to DNA and had a minimum value around 120 nm at the charge ratio of 5. And no significant difference in particle sizes from the charge ratio of 5-20 was found. The suspension of GC/DNA complexes exhibited no significant change in turbidity at the charge ratios of 10, indicating the complete shielding of DNA charge. Cytotoxicity study showed that GC prepared by the water-soluble chitosan had no cytotoxic effects on cells. And transfection efficiency into HepG2, which has asialoglycoprotein receptors (ASGP-R), was higher than that into HeLa without ASGP-R.     

球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化

背景：微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。 方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。 结果与结论：两组培养的细胞数量均呈现前3 d增长,在培养第3天细胞数量达到最高值;实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05),实验组各样本之间细胞数差异无显著性意义(P > 0.05)。倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,培养第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。结果说明,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Antibodies to Haemophilus influenzae type b polysaccharide affect bacterial adherence and multiplication.

Since immunization of infants with conjugated Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) vaccines results in a reduction of colonization, we determined the inhibitory effect of anti-Hib PS on two steps in the colonization, i.e., adherence of H. influenzae to nasopharyngeal epithelium and bacterial growth. Monoclonal antibody (MAb) E117-5 specific for Hib PS inhibited at a concentration of at least 80 microg/ml in vitro the adherence of Hib strain 770235f+b+ to oropharyngeal epithelial cells by 50% (P <, 0.02), but this MAb and sera from children immunized with Hib PS conjugate vaccine (n = 10) were not inhibitory in final dilutions containing up to 20 microg of anti-Hib PS per ml. The growth of Hib strain 770235f+b+ did completely stop in the presence of 5 microg of anti-Hib PS MAb E117-5 per ml and human sera with an anti-Hib PS concentration of 2 microg/ml or more, in contrast to the growth of the nonencapsular variant strain 770235f+b0.     

Morphology and metabolism of Ba-alginate-encapsulated hepatocytes with galactosylated chitosan and poly(vinyl alcohol) as extracellular matrices

—Lactobionic acid, bearing a β-galactose group, was coupled with chitosan to provide synthetic extracellular matrices together with poly(vinyl alcohol) (PVA). The hepatocytes encapsulated in Ba-alginate capsules with galactosylated chitosan (GC) and PVAas extracellular matrices showed aggregation morphologies as the incubation time increased. Ba-alginate-encapsulated hepatocytes with GC exhibited a higher metabolic function in albumin secretion compared to those entrapped in Ba-alginate beads and monolayer-cultured on a collagen-immobilized polystyrene dish. The ammonia removal ability of monolayer-cultured hepatocytes decreased with increasing culture time and disappeared completely after three days. In contrast, the ammonia removal ability of encapsulated and entrapped hepatocytes increased with increasing incubation time in the first seven and five days, respectively. Thereafter, the entrapped hepatocytes lost ammonia removal ability quickly while the encapsulated hepatocytes kept a relatively high ammonia removal ability up to 13 days. The trace amount of GC in the core matrices enabled encapsulated cells to enhance their ammonia removal and albumin secretion ability. The results obtained with 3-(3,4-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) also showed that the capsules incorporated with GC can provide a better microenvironment for cell aggregation along with nutrition and metabolite transfer. Due to the nature of the liquid core, the encapsulated hepatocytes showed very good mobility. This facilitated cell–cell interaction and cell–matrix interaction.     

Tumor necrosis factor-alpha acts as a complete mitogen for primary rat hepatocytes

The cytokine tumor necrosis factor (TNF)-alpha has previously been shown to prime hepatocytes to a state of replicative competence, but has not been shown to act as a complete mitogen for these cells. In the present study we have altered our previously described long-term dimethyl sulfoxide culture system to exclude all known hepatocyte mitogens from the culture media and enable us to directly examine the effects of TNF-alpha on primary rat hepatocytes. We have shown that cells maintained under these culture conditions retain the biochemical and morphological features of well-differentiated hepatocytes. Treatment with TNF-alpha induced DNA synthesis relative to control, to a level not significantly different from that induced by the known hepatocyte mitogen, epidermal growth factor (EGF). Maximal DNA synthesis was induced by treatment with 250 U/ml TNF-alpha for 24 hours. Mitotic figures were observed in cultures treated with TNF-alpha or EGF but not in untreated controls. Treatment of cultures with TNF-alpha, but not EGF, induced activation of both nuclear factor-kappaB p50 homodimers and p50/p65 heterodimers. DNA synthesis induced by TNF-alpha was inhibited by treatment with transforming growth factor-beta. Based on the results of our studies, we conclude that TNF-alpha acts as a complete mitogen for rat hepatocytes.     

Difference in the attachment of hepatocytes between a poly(gamma-benzyl L-glutamate) (PBLG)/poly(N-isopropylacrylamide) (PNIPAAm) diblock copolymer cast surface and a PBlg/PNIPAAm Langmuir-Blodgett one

The effects of temperature on the monolayer behavior of the poly(gamma-benzyl L-glutamate) (PBLG)/poly(N-isopropylacrylamide) (PNIPAAm) diblock copolymer at the air-water interface were examined. Differences in the adhesion and morphology of hepatocytes between Langmuir-Blodgett (LB) films and cast surfaces of the PBLG/PNIPAAm diblock copolymer were investigated. The surface pressure (pi)-area (A) curve of the block copolymer had a tendency to expand with the temperature, due to a change in the conformation of PNIPAAm with the temperature change. Attachment of hepatocytes onto the PBLG/PNIPAAm block copolymer LB surface decreased slightly with an increase of the PNIPAAm content in the block copolymer, whereas that onto the cast surface decreased rapidly with an increase of the PNIPAAm content, due to the hydrophilic property of PNIPAAm in the microphase-separated structure. Rapid morphological changes of the hepatocytes adhered to the LB surfaces, from round shapes to spreading ones, were observed, compared with the cast films. The hepatocytes that adhered to the block copolymer LB surfaces showed less flattened and spread shapes than those that adhered to the PBLG one. Also, the spheroid formation of the hepatocytes increased with an increase of the PNIPAAm content in the block copolymer cast films.     

Fibrinogen and von Willebrand factor mediated platelet adhesion to polystyrene under flow conditions

The roles of adsorbed fibrinogen (Fg) and von Willebrand factor (VWF) in mediating platelet adhesion to synthetic surfaces under flow were investigated using polystyrene (PS) as a model hydrophobic surface. We measured platelet adhesion to PS pre-adsorbed with Fg, VWF, normal plasma, afibrinogenemic plasma, VWF-deficient plasma and deficient plasmas with various concentrations of added Fg or VWF. Platelets in a red blood cell suspension were passed through a flow chamber at either low (50 or 100 s(-1)) or high (500 or 1000 s(-1)) shear. Adhesion to PS pre-adsorbed with afibrinogenemic plasma was very low under both low and high shear conditions, but was restored in a dose-dependent manner with addition of Fg. Less than 20 ng/cm(2)of adsorbed Fg was sufficient to support full-scale platelet adhesion under flow. At high shear rate, platelet adhesion on PS pre-adsorbed with VWF-deficient plasma was much less than on PS pre-adsorbed with normal plasma, but adhesion to PS pre-adsorbed with VWF-deficient plasma with added VWF was very similar to adhesion to PS pre-adsorbed with normal plasma. At low shear, adhesion to PS pre-adsorbed with VWF-deficient plasma was the same as on PS pre-adsorbed with normal plasma. As little as 1 ng/cm(2) of VWF adsorbed from plasma made platelet adhesion higher under high shear than under low shear. The effects of adsorbed Fg and VWF on the morphologies of platelets that adhered from suspensions flowing at high shear rates were also investigated. The lack of either Fg or VWF resulted in marked decreases in the extent of platelet spreading. Real-time observation of platelet adhesion under an epifluorescent microscope showed that platelets adhered to the surface in a linear pattern aligned in the direction of flow under high shear conditions.     

Difference in the attachment of hepatocytes between a poly(γ-benzyl L-glutamate) (PBLG)/poly(N-isopropylacrylamide) (PNIPAAm) diblock copolymer cast surface and a PBLG/PNIPAAm Langmuir-Blodgett one

The effects of temperature on the monolayer behavior of the poly(γ-benzyl L-glutamate) (PBLG)/poly(N-isopropylacrylamide) (PNIPAAm) diblock copolymer at the air-water interface were examined. Differences in the adhesion and morphology of hepatocytes between Langmuir-Blodgett (LB) films and cast surfaces of the PBLG/PNIPAAm diblock copolymer were investigated. The surface pressure (π)-area (A) curve of the block copolymer had a tendency to expand with the temperature, due to a change in the conformation of PNIPAAm with the temperature change. Attachment of hepatocytes onto the PBLG/PNIPAAm block copolymer LB surface decreased slightly with an increase of the PNIPAAm content in the block copolymer, whereas that onto the cast surface decreased rapidly with an increase of the PNIPAAm content, due to the hydrophilic property of PNIPAAm in the microphase-separated structure. Rapid morphological changes of the hepatocytes adhered to the LB surfaces, from round shapes to spreading ones, were observed, compared with the cast films. The hepatocytes that adhered to the block copolymer LB surfaces showed less flattened and spread shapes than those that adhered to the PBLG one. Also, the spheroid formation of the hepatocytes increased with an increase of the PNIPAAm content in the block copolymer cast films.     

壳聚糖球形多孔微载体支持下人肝细胞L-02的高浓度细胞培养

背景：微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。 方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,并对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。 结果：两组培养的细胞数量均呈现前3 d增长,在第3天细胞数量达到最高值;而且实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05),实验组各样本之间差异无显著性意义(P > 0.05);倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。提示,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。     

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.     

Effect of synthesis temperature of PEO-grafted PU/PS IPNs on surface morphology and in vitro blood compatibility

When hydrophilic/hydrophobic polymers have a microdomain structure, platelet adhesion and activation are effectively suppressed by prohibition of the excessive assembly of glycoproteins and adenosine triphosphate (ATP) consumption of the platelets on the surface. In this study, poly(ethylene oxide)-grafted hydrophilic polyurethane (PU)/hydrophobic polystyrene (PS) interpenetrating polymer networks (IPNs) were synthesized by varying the synthesis temperature to control the phase separation and the microdomain surface structure, and the effect of the degree of phase separation on the in vitro blood compatibility. The size of the dispersed PS-rich domains in the PU-rich matrix decreased, and the hydrophilicity also decreased as the synthesis temperature of the PS network during the IPN synthesis was decreased, as the phase separation was suppressed during the synthesis. The amount of the adsorbed bovine plasma fibrinogens (BPF) on the PEO-grafted PU/PS IPNs decreased as the synthesis temperature was decreased, and the in vitro adhesion of the platelets was also suppressed on the PEO-grafted PU/PS IPNs prepared at lower temperature. The microdomain structure on the surface affected the adhesion and the activation of the adhered platelets, and the suppression of the phase separation resulted in the decrease of the domain size, which also enhanced the blood compatibility of the PEO-grafted PU/PS IPNs.     

Effects of EGF and TGFbeta1 on c-myc gene expression and DNA synthesis in embryonic hamster palate mesenchymal cells

Previous work has shown that cell proliferation is a major contributor to the early palate morphogenesis in mammals. The present study was undertaken to examine the effect of EGF, TGFbeta1 and their combination on proliferation (measured by DNA synthesis) and on the expression of a growth related proto-oncogene, c-myc, in embryonic hamster palate mesenchymal cells (HPMC). Vertically developing hamster palatal shelves were dissected on day 11 of gestation, and trypsinized, and primary cultures were grown in DMEM + 10% serum at 37 degrees C and 5% CO2. Following appropriate growth factor treatment of HPMC, DNA synthesis was measured by scintillation counting and extracted RNA was subjected to Northern blot analysis. In serum-starved, pre-confuent cultures treated with EGF (20 ng/ml), DNA synthesis was stimulated in the presence of 2.5% serum. In contrast, treatment of HPMC with TGFbeta1 (10 ng/ml) in the presence or absence of EGF/serum for 24 hr, or HPMC pre-treatment with TGFbeta1 (30 min) followed by EGF/serum (24 hr), resulted in an arrest of DNA synthesis. Northern blot analysis of RNA extracted from HPMC showed that as serum-starved, growth-arrested cells progressed through G0 to G1 phase of the cell cycle, following EGF treatment, c-myc was expressed by 1 hr and declined thereafter. In contrast, TGFbeta1 did not support expression of c-myc. Following pre- or co-treatment with TGFbeta1, the EGF +/- serum-induced expression of c-myc was seen between 1 and 6 hr. It appears that EGF-induced expression of c-myc may be involved in advancing the HPMC in G1, and thus may contribute to the onset of DNA synthesis in HPMC. Since co- or pre-treatment with TGFbeta1 did not inhibit EGF/serum induced expression of c-myc, it is possible that growth arresting effect of TGFbeta1 may not be exerted directly through inhibition or blockage of c-myc expression.     

Effect of synthesis temperature of PEO-grafted PU/PS IPNs on surface morphology and in vitro blood compatibility

When hydrophilic/hydrophobic polymers have a microdomain structure, platelet adhesion and activation are effectively suppressed by prohibition of the excessive assembly of glycoproteins and adenosine triphosphate (ATP) consumption of the platelets on the surface. In this study, poly(ethylene oxide)-grafted hydrophilic polyurethane (PU)/hydrophobic polystyrene (PS) interpenetrating polymer networks (IPNs) were synthesized by varying the synthesis temperature to control the phase separation and the microdomain surface structure, and the effect of the degree of phase separation on the in vitro blood compatibility. The size of the dispersed PS-rich domains in the PU-rich matrix decreased, and the hydrophilicity also decreased as the synthesis temperature of the PS network during the IPN synthesis was decreased, as the phase separation was suppressed during the synthesis. The amount of the adsorbed bovine plasma fibrinogens (BPF) on the PEO-grafted PU/PS IPNs decreased as the synthesis temperature was decreased, and the in vitro adhesion of the platelets was also suppressed on the PEO-grafted PU/PS IPNs prepared at lower temperature. The microdomain structure on the surface affected the adhesion and the activation of the adhered platelets, and the suppression of the phase separation resulted in the decrease of the domain size, which also enhanced the blood compatibility of the PEO-grafted PU/PS IPNs.