Primary hepatocytes are commonly used as liver surrogates in toxicology and tissue engineering fields, therefore, maintenance of functional hepatocytes in vitro is an important topic of investigation. This paper sought to characterize heparin-based hydrogel as a three-dimensional scaffold for hepatocyte culture. The primary rat hepatocytes were mixed with a prepolymer solution comprised of thiolated heparin and acrylated poly(ethylene glycol) (PEG). Raising the temperature from 25° to 37°C initiated Michael addition reaction between the thiol and acrylated moieties and resulted in formation of hydrogel with entrapped cells. Analysis of liver-specific products, albumin and urea, revealed that the heparin hydrogel was non-cytotoxic to cells and, in fact, promoted hepatic function. Hepatocytes entrapped in the heparin-based hydrogel maintained high levels of albumin and urea synthesis after three weeks in culture. Because heparin is known to bind growth factors, we incorporated hepatocyte growth factor (HGF) -an important liver signaling molecule -into the hydrogel. HGF release from heparin hydrogel matrix was analyzed using enzyme linked immunoassay (ELISA) and was shown to occur in a controlled manner with only 40% of GF molecules released after 30 days in culture. Importantly, hepatocytes cultured within HGF-containing hydrogels exhibited significantly higher levels of albumin and urea synthesis compared to cells cultured in the hydrogel alone. Overall, heparin-based hydrogel showed to be a promising matrix for encapsulation and maintenance of difficult-to-culture primary hepatocytes. In the future, we envision employing heparin-based hyrogels as matrices for in vitro differentiation of hepatocytes or stem cells and as vehicles for transplantation of these cells.

An injectable, heparin-based hydrogel system with the potential to be gelled with cells was developed. First, heparin was modified to have thiol groups by the modification of carboxylic groups of heparin with cysteamine using carbodiimide chemistry. Thiol functionalization of heparin carboxylic groups was controlled from 10% to 60% of the available COOH groups, and the retained bioactivity of the modified heparin, characterized by its binding affinity to antithrombin III, decreased with increasing functionalization. Then, the thiol-functionalized heparin was reacted with poly(ethylene glycol) diacrylate to form a hydrogel. The gelation kinetics and mechanical properties of the final gel state could be tuned by controlling cross-link density. Fibroblast cell encapsulation using this hydrogel revealed the nontoxicity of the present system. Cell proliferation inside the hydrogel was observed, and it was significantly enhanced (more than 5-fold) by the addition of fibrinogen into the hydrogel during gelation.

Zerumbone (ZER), present in subtropical ginger Zingiber zerumbet Smith, possesses anti-growth and anti-inflammatory properties in several human cancer cell lines. ZER also down-regulates the cyclooxygenase-2 and inducible nitric oxide synthase expression via modulation of nuclear factor (NF)-jB activation in cell culture systems. These findings led us to investigate whether ZER is able to inhibit carcinogenesis in the colon and lung, using 2 different preclinical mouse models. In Exp. 1, a total of 85 male ICR mice were initiated using a single intraperitoneal (i.p.) injection with azoxymethane (AOM, 10 mg/kg bw) and promoted by 1.5% dextran sulfate sodium (DSS) in drinking water for 7 days for rapid induction of colonic neoplasms. Animals were then fed the diet containing 100, 250 or 500 ppm ZER for 17 weeks. In Exp. 2, a total of 50 female A/J mice were given a single i.p. injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (10 lmol/mouse) to induce lung proliferative lesions. They were then fed the diet mixed with 100, 250 or 500 ppm ZER for 21 weeks. At the termination of the experiments (wk 20 of Exp. 1 and wk 22 of Exp. 2), all animals were subjected to complete necropsy examination to determine the pathological lesions in both tissues. Oral administration of ZER at 100, 250 and 500 ppm significantly inhibited the multiplicity of colonic adenocarcinomas. The treatment also suppressed colonic inflammation. In the lung carcinogenesis, ZER feeding at 250 and 500 ppm significantly inhibited the multiplicity of lung adenomas in a dose-dependent manner. Feeding with ZER resulted in inhibition of proliferation, induction of apoptosis, and suppression of NFjB and heme oxygenase (HO)-1 expression in tumors developed in both tissues. Our findings suggest that dietary administration of ZER effectively suppresses mouse colon and lung carcinogenesis through multiple modulatory mechanisms of growth, apoptosis, inflammation and expression of NFjB and HO-1 that are involved in carcinogenesis in the colon and lung. ' 2008 Wiley-Liss, Inc.Key words: zerumbone; colon; lung; carcinogenesis; chemoprevention A sesquiterpenoid, zerumbone (ZER), is a major constituent of the subtropical ginger plant Zingiber zerumbet Smith. The essential oil of the rhizomes contains large amount of ZER and is used as an anti-inflammatory medicine. 1 Recent studies revealed several biological properties of ZER that may be responsible for inhibition of carcinognesis. They include suppression of skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus activation in Raji cells, 1 inhibition of free radical generation, inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, inhibition of tumor necrosis factor (TNF)-a-release in activated leukocytes and induction of apoptosis in human colonic adenocarcinoma (ADC) cell lines. 2 In vivo studies demonstrated that dietary feeding with ZER markedly suppressed dextran sulfate sodium (DSS)-induced acute colitis in mice 3 and a putat...

In the liver, hepatocytes are exposed to a large array of stimuli that shape hepatic phenotype. This in vivo microenvironment is lost when hepatocytes are cultured in standard cell cultureware, making it challenging to maintain hepatocyte function in vitro. Our article focused on one of the least studied inducers of the hepatic phenotype-the mechanical properties of the underlying substrate. Gel layers comprised of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA) were formed on glass substrates via a radical mediated thiol-ene coupling reaction. The substrate stiffness varied from 10 to 110 kPa by changing the concentration of the precursor solution. ELISA analysis revealed that after 5 days, hepatocytes cultured on a softer heparin gel were synthesizing five times higher levels of albumin compared to those on a stiffer heparin gel. Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype. Our findings point to the importance of substrate mechanical properties on hepatocyte function.

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Matrix metalloproteinase (MMP)-7 (matrilysin-1) plays significant roles in the growth, invasion, and metastasis of colorectal tumors, while (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol with chemopreventive properties, has been shown to be an inhibitor of MMP-2 and MMP-9. In the present study, HT-29 human colorectal cancer cells were treated with EGCG to examine its effects on pro-MMP-7 induction and production using RT-PCR and western blot analyses. Surprisingly, EGCG (10-100 microM) treatment increased both intracellular and extracellular pro-MMP-7 protein levels (2.6-8.4-fold and 1.9-6.4-fold, respectively) in dose- and time-dependent manner, with a significant upregulation of its mRNA expression. EGCG also activated extracellular signal-regulated protein kinase (ERK)1/2, c-JUN NH2-terminal kinase (JNK)1/2 and p38 mitogen-activated protein kinase (MAPK), as previously reported. In addition, the polyphenol triggered the phosphorylation of c-JUN (Ser63 and Ser73) and induced c-JUN/c-FOS, thereby increasing the DNA binding activity of activator protein-1 (AP-1), as shown by an AP-1 luciferase reporter assay. Pharmacological blockade of MAPK activities suggested that pro-MMP-7 expression was induced via JNK1/2 activation, but not in the case of ERK1/2 or p38 MAPK. N-Acetyl-L-cysteine, superoxide (O2-) dismutase and catalase attenuated the EGCG-induced pro-MMP-7 production, suggesting an involvement of oxidative stress in these events. Conversely, EGCG spontaneously generated O2- in a cell-free system that utilized a cytochrome C reduction method. Further, (-)-epicatechin-3-gallate (25 and 100 microM) and green tea polyphenols (33 and 132 microg/ml) induced pro-MMP-7 expression, whereas (-)-epicatechin and (-)-epigallocatechin (100 microM each) did not. Induction of pro-MMP-7 expression by EGCG was also shown in another human colorectal adenocarcinoma cell line, Caco-2. Our results suggest that some green tea catechins induce pro-MMP-7 production via O2- production and the activation of JNK1/2, c-JUN, c-FOS and AP-1 in HT-29 cells.

Delivering isolated chondrocytes with matrix is a promising approach to promote the cartilage repair. The present study attempted to combine the advantages of porous scaffold and hydrogel in delivering chondrocytes to partial-thickness cartilage defects. An electrospun, gelatin-incorporated PLCL scaffold mechanically similar to natural cartilage was fabricated, and chondrocytes were seeded using an injectable heparin-based hydrogel for efficient cell seeding. The scaffold/hydrogel composite showed more enhanced expression of chondrogenic genes and production of GAGs than those prepared without hydrogel. In addition, significant cartilage formation showing good integration with surrounding, similar to natural cartilage, was observed by scaffold/hydrogel composite system in partial-thickness defects of rabbit knees while no regeneration was observed in control defects. Although no exogenous chondrogenic factors were added, it was evident that the scaffold/hydrogel composite system was highly effective and better than the scaffold alone system without hydrogel for cartilage regeneration both in vitro and in vivo.

An in situ heparin-based forming hydrogel that cures under visible-light is formulated using eosin Y as a photoinitiator with triethanolamine as an electron donor to initiate reaction of thiolated-heparin with acrylate-ended poly(ethylene glycol). Formulations and irradiation conditions are presented for control of heparin content (1.6 to 3.3% w/v), modulus (100–10 000 Pa), and gelation time (30–600 s). Encapsulation of 3T3 fibroblasts in the hydrogel gave over 96% viability for all conditions examined. In vitro characterization of epidermal growth factor released from the hydrogel confirmed that the growth factor remains bioactive. The ability to deliver growth factors, fast gelation kinetics under visible light, and independent control of physical and biochemical properties makes this system a promising candidate for use in regenerative medicine. In particular, irradiation conditions that achieve gelation in 150s are compatible with the stringent light exposure limits of the retina, which affords a wide safety margin for use with other tissues.