Endothelial cell growth factor derived from a human glioblastoma cell line and possible association with tumor angiogenesis

The cytosol of a human glioblastoma cell line (KNS-42) stimulated the growth of both bovine aortic endothelial cells and smooth muscle cells in a dose-dependent manner. The endothelial cell growth activity eluted at an apparent molecular weight of about 30,000 on a Sephadex G-100 column and bound to a heparin-Sepharose column with high affinity to elute at 1.3-1.7 M NaCl. The growth activity was destroyed by heating at 56 degrees C for 30 min, but not by exposure to trypsin, deoxyribonuclease or ribonuclease at 37 degrees C for 30 min. As this factor stimulated the growth of vascular endothelial cells and smooth muscle cells, and vasoproliferative responses in chick embryo chorioallantoic membranes were apparent, this factor may possibly be related to tumor angiogenesis.     

Cryopreservation of collagen-based tissue equivalents. II. Improved freezing in the presence of cryoprotective agents

In Part I of this study we determined an optimal cooling rate for cryopreservation of collagen-based tissue equivalents (TEs) that preserves both the postthaw cell viability and mechanical properties, but results in tissue contraction and an overall loss of opacity. The empirically determined optimal cooling rate (5 degrees C/min) was obtained in a freezing medium consisting solely of phosphate-buffered saline (PBS) at physiological concentration (1x). In the present study we report the effect of freezing on TEs in the presence of PBS and two cryoprotective agents (CPAs) (glycerol and dimethyl sulfoxide [Me(2)SO]), at two different concentrations (0.5 and 1.0 M), to two different end temperatures (-80 and -160 degrees C), at a cooling rate of 5 degrees C/min. The controlled rate freezing experiments, postthaw cell viability, and mechanical property measurements were performed as described in Part I of this study. In addition to studying the effect of CPAs on the postthaw properties of TEs, we also investigated (1). the effect of freezing TEs attached to the substrate (as opposed to detached and floating in medium) to determine differences when freezing TEs subject to static mechanical stress via a mechanical constraint to contraction; (2). the effect of freezing glutaraldehyde-fixed TEs to determine differences in freezing-mediated damage to the microstructure; and (3). the effect of freezing more mature TEs that were incubated for 4 weeks in growth factor-supplemented medium as opposed to 2 weeks in basal medium. All TEs frozen at 5 degrees C/min to -80 degrees C in the presence of 0.5 M glycerol or Me(2)SO in PBS were found to be optimally cryopreserved in terms of maintaining opacity and structure as well as cell viability and mechanical properties as compared with unfrozen TEs. The postthaw mechanical properties were adversely affected by freezing to the lower end temperature of -160 degrees C in the presence of CPAs, with the samples frozen in the 1.0 M concentration of CPAs exhibiting a total loss of structural integrity on thawing. Furthermore, TEs frozen attached to the substrate showed decreased opacity and significant contraction as compared with TEs frozen detached from the substrate, as did cross-linked samples frozen without CPA.     

Measurement of the chondrocyte membrane permeability to Me2SO,glycerol and 1,2-propanediol

The addition of cryopreservative agents (CPAs) to chondrocytes and natural and engineered cartilage is critical to protect the cells and tissues from freezing damage during cryopreservation, but this may cause cell damage, e.g. by osmotic shock. The damage could be minimized by the control of the cell volume excursion with the knowledge of cell membrane permeability. In this study, the cell volume responses of chondrocytes to three commonly used CPAs were evaluated using a perfusion microscope stage. The osmotic response of chondrocytes was measured to the perfusion with 1.4 M dimethyl sulfoxide (Me2SO), 1,2-propanediol and glycerol at 21 degrees C. Cell volumes and their transients were determined with image analysis. The cell membrane permeability parameters, including the hydraulic conductivity (Lp), the CPA permeability (omega) and the reflection coefficients (sigma) in the Kedem-Katchalsky (K-K) model, and the Lp and omega in the two-parameter model were determined. The correlated K-K parameters at 21 degrees C were Lp=0.166 +/- 0.035, 0.149 +/- 0.061, 0.212 +/- 0.041 microm/min atm, omega=(7.630 +/- 0.174) x 10(-2), (1.428 +/- 0.627) x 10(-2), (2.744 +/- 0.775) x 10(-2) microm/s and sigma=0.91 +/- 0.09, 0.82 +/- 0.11, 0.88 +/- 0.10 for Me(2)SO, glycerol and 1,2-propanediol, respectively. For the two-parameter model, the parameter values were Lp=0.163 +/- 0.040, 0.128 +/- 0.031, 0.169 +/- 0.025 microm/min atm, omega=(7.881 +/- 0.178) x 10(-2), (1.529 +/- 0.525) x 10(-2), (3.716 +/- 0.493) x 10(-2) microm/s for Me2SO, glycerol and 1,2-propanediol, respectively. No significant difference in the predictions of cell volume excursion during CPA addition was observed when using either the K-K model or the two-parameter model and it was hence advised to adopt the simple two-parameter model in the evaluation. The measured parameters can be used to optimise the CPA addition and removal protocols to maximize the cell survival during cryopreservation.     

Rapidly cooled human sperm: no evidence of intracellular ice formation

BACKGROUND: The cellular damage that human spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. Alternatively, the cell damage may be the result of an osmotic imbalance encountered during thawing. This article examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. METHODS: In this study, human spermatozoa were cooled at a range of cooling rates from 0.3 to 3000 degrees C/min. The ultrastructure of the samples was examined by cryo scanning electron microscopy and freeze substitution to determine whether intracellular ice formed during rapid cooling and to examine alternative mechanisms of cell injury during rapid cooling. RESULTS: No intracellular ice formation was detected at any cooling rate. Freeze substitution of cells that had been cooled at 3000 degrees C/min and then slowly warmed showed that the cells had become plasmolysed and had evidence of membrane damage. CONCLUSIONS: Cell damage to human spermatozoa, at cooling rates of up to 3000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.     

Ultrastructural and functional studies of cryopreserved rat lungs for transplantation using a new hyperosmolal solution

One of the most promising approaches for extending the period of tissue or organ preservation is to induce a state of cryopreservation. In this report, to achieve successful lung cryopreservation, we created a new hyperosmolal solution (HOS) containing 10% glycerol and 5% dimethyl sulfoxide (Me2SO). We compared the efficacy of HOS with that of Euro-Collins solution (ECS) in rat lung cryopreservation from an ultrastructural and functional point of view. Using ECS, widespread damage to the air-blood barrier was observed even in the lungs cryopreserved at -10 degrees C. At -196 degrees C, the capillary lumen was obstructed with a mesh-like formation derived from deteriorated endothelial cells. In contrast, using HOS, the cryopreserved lungs at -10 degrees C were characterized by well preserved endothelial cells and basal laminae, despite the existence of focal cytoplasmic swelling of epithelial cells. The endothelial cells and basal laminae were successfully preserved up to -196 degrees C. Considering the survival rates at day 7 after cryopreserved lung transplantation, the grafted left lungs functioned normally only when perfused by and preserved in HOS at -10 degrees C. These findings demonstrate that HOS was effective not only for endothelial cells and basal laminae up to -196 degrees C, but also lung cryopreservation at least up to -10 degrees C.     

Perfusion bioreactor for small diameter tissue-engineered arteries

A scaleable perfusion bioreactor has been developed for tissue engineering of small diameter arterial constructs. This modular bioreactor allows for dynamic sequential seeding of smooth muscle and endothelial cells, biomechanical stimulation of cells during culture, and monitoring of tissue growth and maturation. Bovine aortic smooth muscle and endothelial cells were seeded onto porous tubular poly(glycolic acid) nonwoven scaffolds and cultured in the bioreactor under pulsatile flow conditions for up to 25 days. Cell proliferation was more than 3-fold after 4 days, smooth muscle cells expressed differentiated phenotype after 16 days, and collagen and elastin were distributed throughout the construct after 25 days of culture. In bioreactor experiments in which the construct lumen was seeded with endothelial cells by perfusion after 13 days of smooth muscle cell culture, endothelial cell seeding efficiency was 100%, and a confluent monolayer was observed in the lumen within 48 h. These data demonstrate that this perfusion bioreactor supports sequential seeding of constructs with smooth muscle and endothelial cells. Dynamic culture under pulsatile flow leads to cellular expression of differentiated function and extracellular matrix deposition toward the development of tissue-engineered arterial constructs.     

T-2 toxin effect on rat aorta: cellular changes in vivo and growth of smooth muscle cells in vitro

Rats were injected intraperitoneally with T-2 toxin and their aortas were studied by light and electron microscopy. The growth of smooth muscle cell explants taken from the tunica media of aortas of similarly treated animals was observed. A single large dose (2 mg/kg) or four injections of 0.3 mg/kg T-2 toxin caused damage and occasional necrosis of endothelial cells, accumulation of basement membrane-like material in the intima, and swelling and activation of smooth muscle cells in the tunica media. Three or more weeks after the last injection of 0.3 mg/kg T-2 toxin the endothelial cells were normal but an excess of fragmented intimal basement membrane-like material persisted and smooth muscle cells were still activated. Outgrowths from explants of aortic tunica media taken within 1 week of the last dose of T-2 toxin showed marked inhibition of smooth muscle cell growth. Three or more weeks after the toxin, the explants showed significantly increased outgrowths. These findings suggest that T-2 toxin causes early endothelial and smooth muscle cell injury accompanied by inhibition of smooth muscle cell growth in culture. This is followed by stimulation of the proliferative capacity of smooth muscle cells in vitro. If a similar mechanism is operative in vivo, it could explain the chronic vascular changes observed after limited exposure to T-2 toxin.     

Valvular endothelial cells regulate the phenotype of interstitial cells in co-culture: effects of steady shear stress

Valvular endothelial cells interact with interstitial cells in a complex hemodynamic and mechanical environment to maintain leaflet tissue integrity. The precise roles of each cell type are difficult to ascertain in a controlled manner in vivo. The objective of this study was to develop a three-dimensional aortic valve leaflet model, comprised of valvular endothelium and interstitial cells, and determine the cellular responses to imposed lumenal fluid flow. Two leaflet models were created using type I collagen hydrogels. Model 1 contained 1 million/mL porcine aortic valve interstitial cells (PAVICs). Model 2 added a seeding of the lumenal surface of Model 1 with approximately 50,000/cm(2) porcine aortic valve endothelial cells (PAVECs). Both leaflet models were exposed to 20 dynes/cm(2) steady shear for up to 96 h, with static constructs serving as controls. Endothelial cell alignment, matrix production, and cell phenotype were monitored. The results indicate that PAVECs align perpendicularly to flow similar to 2D culture. We report that PAVICs in model 1 express vimentin strongly and alpha-smooth-muscle actin (SMA) to a lesser extent, but SMA expression is increased by shear stress, particularly near the lumenal surface. Model 1 constructs increase in cell number, maintain protein levels, but lose glycosaminoglycans in response to shear. Co-culture with PAVECs (Model 2) modulates these responses in both static and flow environments, resulting in PAVIC phenotype that is more similar to the native condition. PAVECs stimulated a decrease in PAVIC proliferation, an increase in protein synthesis with shear stress, and reduced the loss of glycosaminoglycans with flow. Additionally, PAVECs stimulated PAVIC differentiation to a more quiescent phenotype, defined by reduced expression of SMA. These results suggest that valvular endothelial cells are necessary to properly regulate interstitial cell phenotype and matrix synthesis. Additionally, we show that tissue-engineered models can be used to discover and understand complex biomechanical relationships between cells that interact in vivo.     

The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing

A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2. 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85. 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100 degrees C/min) and the numerically predicted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.     

Replication of Chlamydia pneumoniae in vitro in human macrophages, endothelial cells, and aortic artery smooth muscle cells.

Chlamydia pneumoniae has recently been associated with atherosclerotic lesions in coronary arteries. To investigate the biological basis for the dissemination and proliferation of this organism in such lesions, the in vitro growth of C. pneumoniae was studied in two macrophage cell lines, peripheral blood monocyte-derived macrophages, human bronchoalveolar lavage macrophages, several endothelial cell lines, and aortic smooth muscle cells. Five strains of C. pneumoniae were capable of three passages in human U937 macrophages and in murine RAW 246.7 macrophages. Titers were suppressed in both macrophage types with each passage, as compared with growth titers in HEp-2 cells. Both human bronchoalveolar lavage macrophages and peripheral blood monocyte-derived macrophages were able to inhibit C. pneumoniae after 96 h of growth. Eleven C. pneumoniae strains were capable of replicating in normal human aortic artery-derived endothelial cells, umbilical vein-derived endothelial cells, and pulmonary artery endothelial cells. Infection in human aortic artery smooth muscle cells was also established for 13 strains of C. pneumoniae. The in vitro ability of C. pneumoniae to maintain infections in macrophages, endothelial cells, and aortic smooth muscle cells may provide support for the hypothesis that C. pneumoniae can infect such cells and, when infection is followed by an immune response, may contribute to atheroma formation in vivo. More studies are needed to investigate the complex relationship between lytic infection and persistence and the potential for C. pneumoniae to influence the generation of atheromatous lesions.     

The coronary endothelium-derived hyperpolarizing factor (EDHF) stimulates multiple signalling pathways and proliferation in vascular cells

In the present study we determined whether the endothelium-derived hyperpolarizing factor (EDHF), in addition to its acute effects on vascular tone, activates intracellular signalling pathways other than those associated with Ca2+-dependent K+ channels. EDHF was generated by rhythmic distension of porcine coronary arteries under conditions of combined nitric oxide (NO) synthase/cyclo-oxygenase blockade, and the EDHF-containing luminal incubate was applied to cultured human coronary endothelial or smooth muscle cells. In both cell types, the luminal incubate activated tyrosine kinases, the mitogen-activated protein (MAP) kinases, extracellular signal regulated kinases 1 and 2 (Erk1/2) and p38, as well as protein kinase B/Akt. The constituent responsible for Erk1/2 phosphorylation was identified as a cytochrome P450 (CYP) metabolite, as Erk1/2 activation was attenuated by pretreating the EDHF donor with the CYP 2C inhibitor sulfaphenazole as well as by CYP 2C antisense oligonucleotides. Erk1/2 phosphorylation in detector cells was also observed following the transfer of supernatant from cultured endothelial cells treated with the CYP inducer beta-naphthoflavone. The CYP 2C product 11,12-epoxyeicosatrienoic acid (11,12-EET) also activated tyrosine kinases, Erk1/2 and p38 MAP kinase. Overexpression of CYP 2C8 in native porcine coronary artery endothelial cells resulted in an increase in endothelial 11,12-EET production and Erk1/2 phosphorylation compared to that detected in untreated cells or cells transfected with an antisense CYP 2C8. Endothelial cell number was unaffected by transfection with LacZ or CYP 2C8 antisense but was significantly enhanced in cells overexpressing CYP 2C8. These observations indicate that EDHF/11,12-EET is not simply a vasodilator and that its continuous release under pulsatile conditions in vivo may affect vascular cell signalling and proliferation.     

Optimizing the tensile properties of polyvinyl alcohol hydrogel for the construction of a bioprosthetic heart valve stent

Although bioprosthetic heart valves offer the benefits of a natural opening and closing, better hemodynamics, and avoidance of life-long anticoagulant therapy, they nevertheless tend to fail in 10-15 years from tears and calcification. Several authors, including the present ones, have identified the rigid stent as a factor contributing to these failures. The ultimate solution is an artificial heart valve that has mechanical properties that allow it to move in conformity with the aortic root during the cardiac cycle, has superior hemodynamics, is nonthrombogenic, will last more than 20 years, and mitigates the need for anticoagulants. We have identified a polymer, polyvinyl alcohol (PVA) hydrogel, that has mechanical properties similar to soft tissue. The purpose of this research is to match the tensile properties of PVA to the porcine aortic root and to fabricate a stent prototype for a bioprosthetic heart valve with the use of the PVA hydrogel. Specimens of 15% w/w PVA were prepared by processing through 1-6 cycles of freezing (-20 degrees C) at 0.2 degrees C/min freeze rate and thawing (+20 degrees C) at different thawing rates (0.2 degrees C/min and 1 degrees C/min), for different holding times (1 and 6 h) at -20 degrees C. Subsequently tensile tests and stress-relaxation tests were conducted on the specimens. The different holding times at -20 degrees C demonstrated no difference in the result. The slower thawing rate improved the tensile properties but did not produce significant changes on the stress-relaxation properties. The nonlinear stress-strain curve for the PVA after the fourth freeze-thaw cycle matched the porcine aortic root within the physiological pressure range. The stress-relaxation curve for PVA also approximated the shape of the aortic root. The complex geometry of an artificial heart valve stent was successfully injection molded. These results, in combination with other preliminary findings for biocompatibility and fatigue behavior, suggest that PVA hydrogel is a promising biomaterial for implants, catheters, and artificial skin.     

Neural stem cells differentiation ability of human umbilical cord mesenchymal stromal cells is not altered by cryopreservation

Human umbilical mesenchymal stem cells (HUMSCs) have potential therapeutic use in the recovery of central nervous system injury for their ability to differentiate into neural stem cells. However, for transformed HUMSCs to be constantly available for use during surgery a reliable method of cell storage is necessary. The present study aimed to determine whether a simple method of cryopreservation by slow cooling with Me(2)SO had an effect on the proliferation, secretion and differentiation capacities of HUMSCs. These results demonstrate that cryopreservation has no effect on the phenotype, cell cycle, cell proliferation and the ability to secret neurotrophins. Non-cryopreserved and cryopreserved HUMSCs showed the similar ability to differentiate into neural stem-like cells. There results show that cryopreservation by slow cooling with Me(2)SO is effective to retain the proliferation and neural differentiation ability of HUMSCs, cryopreserved HUMSCs maybe very useful for future clinical applications in neural regenerative medicine.     

1H NMR spectroscopy as a tool to evaluate key metabolic functions of primary porcine hepatocytes after cryopreservation

Proton NMR spectroscopy of biological fluids has produced interesting results lately. We used the technique to investigate the effects of cryopreservation on primary porcine hepatocytes as successful cryopreservation of primary porcine hepatocytes is of importance to the development of bioartificial liver support systems. After isolation 10(8) hepatocytes were cryopreserved for 1 week in Williams E/10% DMSO, either by quick freezing (-5 to -30 degrees C/min), slow freezing (-0.3 to -3 degrees C/min) or stepwise freezing protocols on cell suspensions and confluent cell plates. Plating efficiency was assessed by percentage LDH release. Metabolic functions of cryopreserved hepatocytes at 24 h post-thawing were compared with those of fresh hepatocyte cultures at 48 h. 1H nuclear magnetic resonance spectroscopy of the culture medium post-incubation, using the presaturation technique, assessed the following: glucose metabolism, transamination and glutamine synthesis and succinate synthesis. Freshly isolated cells had a viability of 82 +/- 4.3% and a plating efficiency of 87 +/- 3.8%. All cryopreservation protocols resulted in significantly reduced viability and plating efficiency. No significant differences were observed between different cryopreservation media or protocols. When comparing cryopreserved with freshly isolated cells, we observed that metabolism of acetyl-CoA precursors was significantly impaired in cryopreserved cells. Lactate and pyruvate production was also significantly less, although glucose consumption was similar. No differences were observed in gluconeogenic amino acid metabolism, transamination and urea synthesis. 1H NMR spectroscopy can be used to provide information about metabolic activity and functions of cultured primary cells.     

脂肪间充质干细胞分化为内皮细胞并体外构建组织工程心脏瓣膜

背景：组织工程心脏瓣膜是利用组织工程技术将种子细胞种植于瓣膜支架上所构建的一种人工瓣膜,目前国内外研究主要集中于种子细胞来源及支架选择上。 目的：探讨人脂肪间充质干细胞体外向内皮细胞诱导分化后的细胞作为种子细胞,脱细胞猪主动脉瓣膜作为支架体外构建组织工程心脏瓣膜的可行性。 方法：利用吸脂术采集脂肪组织,分离、培养脂肪间充质干细胞,流式细胞仪鉴定细胞表型;免疫细胞化学方法及RT-PCR检测细胞分化标志物;应用Triton X-100联合胰蛋白酶的方法制备脱细胞猪主动脉瓣支架,将体外培养扩增的诱导分化后的内皮细胞种植于支架上构建组织工程心脏瓣膜,光镜及电镜下观察组织工程心脏瓣膜的组织学结构。 结果与结论：脂肪组织分离培养的脂肪间充质干细胞向内皮细胞诱导分化后表达CD31、CD34、CD144、Ⅷ因子和内皮型一氧化氮合成酶等内皮细胞特异性抗原;脱细胞猪主动脉瓣膜支架脱细胞完全,弹力纤维及胶原纤维保持完整;构建的组织工程心脏瓣膜可见支架上排列连续的单细胞层。提示脂肪间充质干细胞在体外向内皮细胞诱导分化后已初步具有内皮细胞功能,在脱细胞猪主动脉瓣膜支架上生长良好,可以在体外初步构建组织工程心脏瓣膜。     

Cryopreservation of collagen-based tissue equivalents. I. Effect of freezing in the absence of cryoprotective agents

The effect of freezing on the viability and mechanical properties of tissue-equivalents (TEs) was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. TEs (a class of bioartificial tissues) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. TEs were detached from the substrate and frozen in phosphate-buffered saline using a controlled rate freezer (CRF) at various cooling rates (0.5, 2, 5, 20, and 40 degrees C/min to -80 or -160 degrees C) or in a directional solidification stage (DSS) (5 degrees C/min to -80 degrees C) or slam frozen (>1000 degrees C/min). Viability of the fibroblasts in the TEs was assessed by ethidium homodimer and Hoechst assays immediately after thawing. Uniaxial tension experiments were also performed on an MTS (Eden Prairie, MN) Micro Bionix system to assess the postthaw mechanical properties of the frozen-thawed TEs. Cooling rates of either 2 or 5 degrees C/min using the CRF were optimal for preserving both immediate cell viability and mechanical properties of the TEs, postthaw. By 72 h postthaw, TEs frozen in the CRF at 5 degrees C/min to -80 degrees C showed a slight decrease in cell viability, with a significant increase in tangent modulus and ultimate tensile stress suggesting a cell-mediated recovery mechanism. Both the postthaw mechanical properties and cell viability are adversely affected by freezing to the lower end temperature of -160 degrees C. Mechanical properties are adversely affected by freezing in the DSS.     

Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses

A multilayered silk fibroin protein coating system was employed as a drug carrier and delivery system to evaluate vascular cell responses to heparin, paclitaxel, and clopidogrel. The results demonstrated that the silk coating system was an effective system for drug-eluting coatings, such as for stent applications, based on its useful micromechanical properties and biological outcomes. Cell attachment and viability studies with human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCASMCs) on the drug-incorporated silk coatings demonstrated that paclitaxel and clopidogrel inhibited smooth muscle cell (SMC) proliferation and retarded endothelial cell proliferation. Heparin-loaded silk multilayers promoted HAEC proliferation while inhibiting HCASMC proliferation, desired outcomes for the prevention of restenosis. The preservation of the phenotype of endothelial cells on silk and heparin-loaded silk coatings was confirmed with the presence of endothelial markers CD-31, CD-146, vWF and VE-Cadherin using immunocytochemistry assays. A preliminary in-vivo study in a porcine aorta showed integrity of the silk coatings after implantation and the reduction of platelet adhesion on the heparin-loaded silk coatings.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells exposed to physiologic shear stress

Hydrogen peroxide (H2O2) is produced by inflammatory and vascular cells and induces oxidative stress, which may contribute to vascular disease and endothelial cell dysfunction. In smooth muscle cells, H2O2 induces production of O2 by activating NADPH oxidase. However, the mechanisms whereby H2O2 induces oxidative stress in endothelial cells are not well understood, although O2 may play a role. Recent studies have documented increased O2 in endothelial cells exposed to H2O2 via uncoupled nitric oxide synthase (NOS) and NADPH oxidase under static conditions. To assess responses to H2O2 in porcine aortic endothelial cells (PAEC) under shearing conditions, a constant flow rate of 24. 4 ml/min was applied to produce physiologically relevant shear stress (8. 2 dynes/cm). Here we demonstrate that treatment with 100 muM H2O2 increases intracellular O2 levels in PAEC. In addition, we demonstrate that l-NAME, an inhibitor of NOS, and apocynin, an inhibitor of NADPH oxidase, reduced O2 levels in PAEC treated with H2O2 under physiologic shear suggesting that both NOS and NADPH oxidase contribute to H2O2-induced O2 in PAEC. Co-inhibition of NOS and NADPH oxidase also reduced intracellular O2 levels under shear. We conclude that H2O2-induced oxidative stress in endothelial cells exhibits increased intracellular O2 levels through NOS and NADPH oxidase under shear. The inhibition of NOS and NADPH with H2O2 exposure is nonlinear, suggesting some interdependent or compensating system within endothelial cells. These findings suggest a complex interaction between H2O2 and oxidant-generating enzymes that may contribute to endothelial dysfunction in cardiovascular diseases.     

Temperature-Responsive surface for novel co-culture systems of hepatocytes with endothelial cells: 2-D patterned and double layered co-cultures

We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintained intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.     

内皮细胞条件培养液对平滑肌细胞合成胶原的影响

本实验采用胶原酶消化法分离、培养兔主动脉内皮细胞(EC)及平滑肌细胞(SMC),以[~3H]-脯氨酸掺入SMC合成的[~3H]-羟脯氨酸中的量作为测定胶原的指标,观察了兔主动脉内皮细胞条件培养液(EC-CM)对动脉SMC合成胶原的影响,结果发现融合的EC-CM能促进SMC的胶原合成,但SMC数无明显变化;1:2稀释的EC-CM促进SMC合成胶原的作用最大。可见EC通过促进SMC的胶原合成而参与了动脉粥样硬化过程。