Growth of human cells on polyethersulfone (PES) hollow fiber membranes

A novel material of porous hollow fibers made of polyethersulfone (PES) was examined for its ability to support the growth of human cells. This material was made in the absence of solvents and had pore diameters smaller than 100 microm. Human cell lines of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were investigated for adherence, growth, spread and survival on PES by confocal laser microscopy after staining of the cells with Calcein-AM. Endothelial cell attachment and growth required pre-coating PES with either fibronectin or gelatin. The other cell types exhibited little difference in growth, spread or survival on coated or uncoated PES. All the cells readily adhered and spread on the outer, inner and cut surfaces of PES. With time confluent monolayers of cells covered the available surface area of PES and in some cases cells grew as multilayers. Many of the cells were able to survive on the PES for up to 7 weeks and in some cases growth was so extensive that the underlying PES was no longer visible. Scanning electron microscope observations of cells on the materials correlated with the confocal morphometric data. Thus, PES is a substrate for the growth of many different types of human cells and may be a useful scaffolding material for tissue engineering.     

Vascularization of hollow channel-modified porous silk scaffolds with endothelial cells for tissue regeneration

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo, the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications.     

Cytocompatibility of poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) membranes with human endothelial cells and macrophages

Polyacrylonitrile modified with N-vinyl-2-pyrrolidone (NVP) shows good hemocompatibility. This work, which aims to evaluate the cytocompatibility of membranes fabricated from poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) (PANCNVP), studied the adhesion of macrophages and endothelial cell (EC) cultures. It was found that PANCNVP membranes with higher NVP content decreased the adhesion of both macrophages and ECs. Compared with polyacrylonitrile and tissue culture polystyrene control, however, these PANCNVP membranes promoted the proliferation of ECs. Furthermore, the viability of ECs cultured on the PANCNVP membrane surfaces was also relatively competitive. Both static and dynamic water contact angle measurements were conducted to explain the nature of cell adhesion to the PANCNVP membranes. On the basis of these results and the phenomena of water swelling and water states reported previously, it was presumed that the coexistence of large amounts of bound water and free water induced by NVP moieties are responsible for the lower adhesion and better function of cells adhering to the PANCNVP membranes.     

Cytokine regulation of env gene expression of human endogenous retrovirus-R in human vascular endothelial cells.

To determine whether human endogenous retroviruses are implicated in the pathogenesis of inflammatory vascular diseases of unknown etiology, we examined mRNA expression of a human endogenous retrovirus, HERV-R, which has a long open reading frame in the env region, in cultured human vascular endothelial and smooth muscle cells stimulated in the presence of various cytokines. mRNA of HERV-R was always evident in these cells but not in fibroblastic cells. Levels of expression in vascular endothelial cells were significantly regulated by treatment with tumor necrosis factor-alpha, interleukin (IL)-1alpha, and IL-1beta as up-regulators and interferon-gamma as a down-regulator. These observations are interpreted to mean that HERV-R expression may be up- or down-regulated at sites of inflammation in vessels in vivo and hence may play a pathogenetic role in inflammatory vascular diseases in humans, perhaps similar to endogenous retroviruses in mouse models of polyarteritis nodosa in humans.     

Role of glycosaminoglycans (GAGs) in regulation of the immunogenicity of human vascular endothelial cells

Heparan sulphate is a common glycosaminoglycan component of proteoglycans present on the luminal surface of vascular endothelium. It has been proposed that an important function of these molecules is the sequestration of a range of proinflammatory and proadhesive cytokines. Such cytokines play a vital role during lymphocyte recruitment from the blood at sites of inflammation. In this study it is shown that the effects of interferon-gamma (IFN-γ), but not of tumour necrosis factor-alpha (TNF-α), are inhibited by treatment with soluble heparin. Specifically, heparin was shown to inhibit the induction of class II MHC antigens and the up-regulation of intercellular adhesion molecule-1 (ICAM-1) produced by treatment of cultured human endothelial cells with IFN-γ. Furthermore, it was shown that heparin blocked the enhanced adhesion of T lymphocytes to IFN-γ-treated endothelial cells. Investigation of the inhibitory effects of other GAG molecules demonstrated a requirement for heparin-like structural domains as chondroitin sulphate was unable to inhibit the function of IFN-γ. These results may explain reported immunosuppressive properties of heparin, and are consistent with the model that heparin may compete with cell surface GAGs to bind IFN-γ, thereby reducing effective biological activity.     

Degradation of hollow mesoporous silica nanoparticles in human umbilical vein endothelial cells

Hollow mesoporous silica nanoparticles (HMSNs) are considered a potential drug delivery system owing to their recognized advantages in drug loading and releasing. However, whether HMSN could be degraded inside the cells remains unknown. In this study, based on the observations by transmission electron microscopy, fluorescence staining, enzymatic proteolysis, and inductively coupled plasma atomic emission spectroscopy, HMSNs were proved to be degradable in human umbilical vein endothelial cells. The degradation first took place in cytoplasm and lysosomes, and secondly in lysosomes only. The Si content in culture medium increased as the time increases, suggesting that the degradation product inside the cells could be excreted into the culture medium. The degrading rate is fast in the first 2 days and slow after 2 days. The present results provided a clue to further research on the metabolic way and cytotoxicity of silica nanoparticles.     

Vascularization of the human adult sternum (author's transl)

The study of the vascularization of the sternum allows to individualise the vascular pedicles, the anterior and posterior peripheral sternal networks and the intrasternal vascularization. 1. The numerous vascular pedicles of the sternum all come from the internal mammary arteries. The postero-superior and postero-inferior sternal arteries of one simple intercostal space or of contiguous spaces are anastomosed by systems of latero-sternal, retroarticular and retro-sternal arches. From these arches come vessels which go directly to the sternum or which form the posterior peripheral sternal network. The anterior sternal branches of perforating arteries form arches or go directly to the anterior peripheral sternal network or to the sternum. The veins are less anastomosed than the arteries and therefore their arches are less distinct. 2. The anterior and posterior peripheral sternal networks are especially developed with respect to the lower half of the body of the sternum at the level of the 4th and 5th intercostal spaces. The posterior network is more developed than the anterior network. The arteries often have a spiral course, their anastomoses occur with their contralateral and homolateral homologues. The veins, which are of larger calibre than the arteries, design a denser and more irregular network. 3. The intra-skeletal vascularization, tributary of the peripheral networks, arches and branches of the internal mammary vessels, includes a network of the compact bone and a medullary network. The network of the compact bone, situated in one plane, is composed of polyhedral elongated meshes, according to the large axe of the sternum. The medullary network may be divided into a sub-cortical and a centro-medullary part. The sub-cortical part is composed of a peripheral zone and a more central zone. The totality of the intramedullary network includes a few fine arteries, an interadipocytary network, which is very developed and is variable in form. It also include sinuses of increasing order connected into the venous system, into the network of the compact bone, or into the large trans-cortical veins, which are drained by the peripheral sternal networks or by the collateral veins or the internal mammary system.     

Characterization and regulation of the urokinase receptor of human endothelial cells

Endothelial cells synthesize and secrete urokinase-type plasminogen activators (u-PA) in response to various stimuli. To modulate the local expression of u-PA activity both intravascularly and pericellularly during angiogenesis, endothelial cells express both inhibitors (primarily PAI-1) and receptors (u-PAR) for u-PA. The interaction of u-PA with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Using radioligand binding studies, we and others have demonstrated that human umbilical vein endothelial cells (HUVEC) express high affinity receptors for urokinase on the cell surface. We have demonstrated that single chain urokinase (scu-PA, prourokinase) binds only via the growth factor domain, while two chain high molecular weight urokinase (tcu-PA) can bind to the receptor or to cell- or matrix-associated PAI-1. We have isolated a ∼46kDa glycoprotein from HUVEC using affinity chromatography which retains the ability to specifically bind u-PA. At least three post -translational modifications appear to occur including removal of an N-terminal signal peptide; N-linked glycosylation, and C-terminal cleavage with addition of a phosphatidylinositol-glycan moiety which links the externally oriented protein to the cell surface. Using the polymerase chain reaction and published sequence information of the u-PAR cloned from a transformed fibroblast cDNA library, we amplified cDNAs of u-PAR from HUVEC and PMA-treated U937 cells. The specificity of the cDNAs was confirmed by restriction mapping and direct sequence analysis. Using these probes and radioligand binding studies we have demonstrated that at least two independent protein kinase pathways exist in endothelial cells for upregulating u-PA receptor expression. Down regulation of receptors may be pathophysiologic in thrombosic disorders whereas up regulation may be important in promoting wound healing, vascular repair, and protection from thrombosis. Up regulation could be harmful as well in such conditions as pathologic neovascularization (e.g., diabetic retinopathy) and in tumour metastasis as well as in tumour-related angiogenesis. Understanding the control and functional significance of u-PA binding to cells in general will hopefully enable the design of therapies to optimize the beneficial aspects and minimize any harmful effects of this interaction. Changes in the local expression of u-PA, PAM and u-PAR by endothelial cells may affect the extent of angiogenesis or the degree of local intravascular fibrinolysis, which might be critical in conditions such as unstable angina and myocardial infarction.     

Morphological studies on the culture of kidney epithelial cells in a fiber-in-fiber bioreactor design with hollow fiber membranes

A hollow fiber-in-fiber-based bioreactor system was tested for the applicability to host kidney epithelial cells as a model system for a bioartificial kidney. Hollow fibers were prepared from polyacrylonitrile (PAN), polysulfone-polyvinylpyrollidinone (PVP) blend (PSU) and poly(acrylonitrile-N-vinylpyrollidinone) copolymer P(AN-NVP). Hollow fibers with smaller and larger diameters were prepared so that the smaller fitted into the larger, with a distance of 50-100 microm in between. The following material combinations as outer and inner fiber were applied: PAN-PAN; PSU-PSU, PSU-P(AN-NVP). Madin-Darby kidney epithelial cells (MDCK) were seeded in the interfiber space and cultured for a period up to 14 days. Light, scanning, and transmission electron microscopy were used to follow the adhesion and growth of cells, and to characterize their morphology. As a result, we found that MDCK cells were able to grow in the interfiber space in mono- and multilayers without signs of systemic degeneration. Comparison of the different materials showed that PAN and P(AN-NVP) provided the best growth conditions, indicated by a tight attachment of cells on hollow fiber membrane, and subsequent proliferation and development of structural elements of normal epithelia, such as tight junctions and microvilli. In conclusion, the fiber-in-fiber design seems to be an interesting system for the construction of a bioartificial kidney.     

Poly(amidoamine) dendronized hollow fiber membranes: synthesis, characterization, and preliminary applications as drug delivery devices

Poly(amidoamine) (PAMAM) dendrons were prepared from hollow fiber membranes (HFM) consisting of bromomethylated poly(2,6-dimethyl-1,4-phenylene oxide) (BPPO) in a stepwise manner. The prepared HFM were characterized by Fourier transform infrared spectroscopy, elemental analysis, and scanning electron microscopy. The drug loading efficiency and release behavior of the PAMAM dendronized HFM were evaluated using sodium salicylate, sodium methotrexate, and Congo red as model drugs. The results suggest that PAMAM dendronized HFM can be effectively loaded with a variety of drugs and prolong the release of these drugs. The drug loading and release characteristics of the HFM depend on the generation of PAMAM dendrons grafted on the membranes. The prepared PAMAM dendronized BPPO HFM are promising scaffolds in drug delivery and tissue engineering.     

Fabrication and characterization of permeable degradable poly(DL-lactide-co-glycolide) (PLGA) hollow fiber phase inversion membranes for use as nerve tract guidance channels

Biodegradable permeable poly(DL-lactide-co-glycolide) (PLGA) hollow fiber membranes (HFMs) were fabricated using a wet phase inversion technique. By varying several parameters, such as the spinneret size, solvent and non-solvent pair, polymer concentration, flow rate, precipitation method, drop height, and small molecular pore-forming agents, PLGA HFMs with variable sizes, surface morphologies, porosities, and diffusive permeability were obtained. Under simulated physiological conditions in vitro, PLGA HFMs exhibited a degradation profile to accommodate nervous system regeneration and axonal outgrowth. While accelerated degradation resulted in substantial molecular weight loss starting at 2 weeks and loss of selective permeability at 3 weeks, PLGA HFMs maintained gross structural integrity in the first 4 weeks, followed by sharp weight loss at 6 weeks and complete disappearance at about 8 weeks. When compared to the raw PLGA material in a pellet form, which underwent heterogeneous degradation, the PLGA HFMs exhibited a homogeneous degradation where the surface and bulk degraded at approximately the same rate, and an overall lower degradation rate. Our results indicate that using a wet phase inversion technique, degradable HFMs with variable size, inner and outer surface morphologies, porosity, and permeability with potential applications for nerve tract guidance conduits can be fabricated.     

Growth of human endothelial cells on photochemically grafted Gly-Arg-Gly-Asp (GRGD) chitosans

To improve ECs adhesion and growth on chitosan, cell adhesive peptide Gly-Arg-Gly-Asp (GRGD) was photochemically grafted to its surface. Grafting 0.025 M of GRGD-SANPAH (N-Succinimidyl-6-[4'-azido-2'-nitrophenylamino]-hexanoate) solutions to chitosan and tripolyphosphate anhydrous crosslinked chitosan (chitosan-TPP) surfaces was performed by surface adsorption of the peptide and subsequent ultraviolet (UV) irradiation for photoreaction. FTIR spectra and electron spectroscopy for chemical analysis (ESCA) confirmed that GRGD was successfully grafted to form chitosan-GRGD surfaces (e.g., the composition fraction of nitrogen (N(1s)) for chitosan-GRGD surface was 5.2% instead of 3.2% for chitosan only). Moreover, the grafting efficiencies for 0.025 M of GRGD to chitosan and chitosan-TPP surfaces were about 83% and 53%, semi-quantitatively analyzed by an HPLC. Human umbilical vein ECs were well adhered and grew on chitosan-GRGD and chitosan-TPP-GRGD surfaces after 36 h of incubation but not chitosan and chitosan-TPP ones. Moreover, the viability for the growth of ECs on the chitosan-GRGD and chitosan-TPP-GRGD surfaces analyzed by MTT assay also confirmed the efficacy of the GRGD immobilization. In conclusion, the chitosan-GRGD and chitosan-TPP-GRGD surfaces were prepared by a photochemical technique that could enhance the adhesion and growth of ECs on those surfaces for further applications for tissue engineering.     

Vascularization of PEG-grafted macroporous hydrogel sponges: a three-dimensional in vitro angiogenesis model using human microvascular endothelial cells

The native extracellular matrix (ECM) of elastic tissues is strong and flexible and supports cell adhesion and enzymatic matrix remodeling. In an attempt to convey these ECM properties to a synthetic scaffold appropriate for soft tissue engineering applications, a biodegradable, elastomeric poly(ester urethane)urea (PEUU) was combined with type I collagen at various ratios (2.5, 5, 10, 20, 50, 60, 70, 80, and 90 wt% collagen) and electrospun to construct elastic matrices. Randomly orientated fibers in the electrospun matrices ranged in diameter from 100-900 nm, dependent on initial polymer concentration. Picrosirius red staining of matrices and CD spectroscopy of released collagen confirmed collagen incorporation and preservation of collagen structure at the higher collagen mass fractions. Matrices were strong and distensible possessing strengths of 2-13 MPa with breaking strains of 160-280% even with low PEUU content. Collagen incorporation significantly enhanced smooth muscle cell adhesion onto electrospun scaffolds. An approach has been demonstrated that mimics elastic extracellular matrices by using a synthetic component to provide mechanical function together with a biomacromolecule, collagen. Such matrices may find application in engineering soft tissue.     

The performance of primary human renal cells in hollow fiber bioreactors for bioartificial kidneys

Bioartificial kidneys (BAKs) containing human primary renal proximal tubule cells (HPTCs) have been applied in clinical trials. The results were encouraging, but also showed that more research is required. Animal cells or cell lines are not suitable for clinical applications, but have been mainly used in studies on BAK development as large numbers of such cells could be easily obtained. It is difficult to predict HPTC performance based on data obtained with other cell types. To enable more extensive studies on HPTCs, we have developed a bioreactor containing single hollow fiber membranes that requires relatively small amounts of cells. Special hollow fiber membranes with the skin layer on the outer surface and consisting of polyethersulfone/polyvinylpyrrolidone were developed. The results suggested that such hollow fiber membranes were more suitable for the bioreactor unit of BAKs than membranes with an inner skin layer. An HPTC-compatible double coating was applied to the insides of the hollow fiber membranes, which sustained the formation of functional epithelia under bioreactor conditions. Nevertheless, the state of differentiation of the primary human cells remained a critical issue and should be further addressed. The bioreactor system described here will facilitate further studies on the relevant human cell type.     

Hydrodynamically stable adhesion of endothelial cells onto a polypropylene hollow fiber membrane by modification with adhesive protein

The effect on the adhesion of endothelial cells of immobilization of adhesion proteins onto a microporous polypropylene hollow fiber membrane for a conventional artificial lung was investigated with the aim of constructing a hybrid artificial lung bearing endothelial cells on the modified membrane. The membrane was modified by adsorption or covalent bonding of adhesion proteins of fibronectin, gelatin, or Pronectin. The density of adherent cells on the membrane modified by adsorption of or covalent bonding with fibronectin reached 1 × 10⁵ cells/cm² after 1 day of incubation, which corresponds to the confluent cell density in a conventional culture dish, while the cell densities on the membranes modifieds with gelatin and Pronectin were 1–5 × 10⁴ cells/cm² and 0.5–1 × 10⁴ cells/cm², respectively. The loading of hydrodynamic shear force (0.23N/m²) for 30min to the membranes bearing endothelial cells had little effect on the density of adhered cells. The membrane covalently bonded with fibronectin could well maintain a high cell density even after the loading of a higher shear force of 1.15N/m² for 180min, however, at this level of shear force 49% of adhered cells on the fibronectin-adsorbed membrane were lost after 30min. A partial cardiopulmonary bypass in rats employing the hybrid artificial lung model composed of a polypropylene hollow fiber membrane covalently bonded with fibronectin and endothelial cell adhesion showed the inhibition of tumor necrosis factor- release and an increase in IL-10 concentration in the circulating blood compared with that employing an artificial lung without cells. Long-term partial cardiopulmonary bypass employing the hybrid artificial lung model should be studied further.     

用小双链RNA抑制转化入HUVEC中绿色荧光蛋白基因的表达

目的：建立稳定表达绿色荧光蛋白(GFP)的人脐静脉血管内皮细胞（HUVECs），研究小干扰RNA(siRNA)对HUVECs中GFP表达的抑制作用。 方法： 用lipofectamine2000将编码GFP的质粒pN3-EGFP转入HUVECs中。用G418筛选并维持已转化pN3-EGFP的HUVEC(HUVEC-GFP)。利用T7 RNA转录试剂盒，制备可抑制GFP基因表达的siRNA（GFPsiRNA）和无关对照的RNA（control siRNA）。用oligofectamine将siRNA转入HUVEC-GFP中。继续培养48 h后，检测HUVEC-GFP中GFP蛋白和mRNA表达水平。 结果： 用G418筛选获得了HUVEC-GFP细胞株，可以观察到GFP的稳定表达。HUVEC-GFP转化siRNA后48 h，GFP的荧光强度明显下降，而对照组的荧光强度无明显下降。半定量RT-PCR检测显示，GFPsiRNA对GFP mRNA表达有较强的抑制作用，抑制率达40%，而control siRNA对GFP mRNA表达水平无明显的抑制作用。 结论： 利用体外转录合成的siRNA能有效地抑制HUVECs中GFP的表达。     

Cytokine regulation of hemostatic property and IL-6 production of human endothelial cells

The effects of four inflammatory cytokines, IL-1, TNF, IFN-, and IL-6, were assessed on the following functions of human vascular endothelial cells (EC) in culture: expression of procoagulant activity (PCA), endothelial cell-associated thrombomodulin (TM), and IL-6 production. Both IL-1 and TNF induced PCA, reduced TM, and induced IL-6 production in a dose-dependent manner. IFN- had a weak but significant reducing effect on TM and an inducing effect on IL-6 production, while it had no effect on PCA expression. IFN-, however, when added in combination with either IL-1 or TNF, modulated the effects of these cytokines; INF- inhibited the PCA expression and enhanced the reduction of TM and the production of IL-6, which were induced by either IL-1 or TNF. In contrast, IL-6 had no significant effect on the EC functions studied. These results suggest that both IL-1 and TNF are the major cytokines affecting the EC functions that determine the association between the coagulation and the inflammatory response, and that IFN- affects this phenomenon predominantly through the modification of the effects of these cytokines.     

内皮细胞中一种新的可溶型黏附分子sCD226的表达调变规律

目的：探讨人内皮细胞中一种新的可溶型黏附分子sCD226的表达调变规律。方法：用ELISA方法，检测不同浓度的LPS刺激人脐静脉内皮细胞后不同时间点培养上清中sCD226含量的变化；用Greiss法检测上述细胞培养上清中N0含量的变化，并分析两者的线性相关性。加入抗TNF-α中和抗体和iNOS抑制剂后，观察其对LPS诱导sCD226生成的影响。结果：①随着LPS刺激剂量的增加和刺激时间的延长，HUVEC培养上清中sCD226的含量明显增加，100mg／L LPSS刺激3d后sCD226的含量为188．5μg／L。②LPS刺激剂量为100mg／L时，HUVEC培养上清中sCD226和N0的含量呈显著的正相关。③抗TNF—α中和抗体和iNOS抑制剂可显著抑制LPS活化的HUVEC产生sCD226。结论：LPS可诱导HUVEC产生sCD226分子，且sCD226的生成与N0和TNF-α的产生密切相关。