Sabrina Morelli

Evaluation of cell behaviour related to physico-chemical properties of polymeric membranes to be used in bioartificial organs.

In bioartificial organs using isolated cells, polymeric semipermeable membranes are used as immunoselective barriers as a means for cell oxygenation and also as substrata for adhesion of anchorage-dependent cells. The capacity of the membrane to perform its functions and to provide a cytocompatible support for cell culture depends in particular on its surface properties. In this study we investigated the physico-chemical aspects of the interaction between the membrane and mammalian cells in order to provide guidelines to the selection of cytocompatible membranes. We evaluated the adhesion and metabolic behaviour of isolated liver cells cultured on various polymeric membranes such as those modified by protein adsorption. The physico-chemical properties of the membranes were characterised by contact angle measurements. The different parameters such as acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) of the surface free energy were calculated according to Good-van Osss model. The adsorption of protein modified markedly both contact angle and components of membrane surface tension. In particular, base parameter of surface tension decreased drastically with increased water contact angle. For each investigated membrane we observed that cell adhesion increased with increasing base parameter of membrane surface tension. The absolute value of cell adhesion is higher in the presence of serum proteins adsorbed on the membrane surface, which change the wettability by increasing the base parameter of surface tension. Also, the metabolic functions improve on hydrophilic membranes. Liver cells synthesised urea with a rate that increased with increasing base parameter value of membrane surface tension. The metabolic activity is particularly expressed at high levels when cells were cultured on polycarbonate and cellulose acetate membranes.

Human hepatocyte functions in a crossed hollow fiber membrane bioreactor.

An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour. A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 microg/h 10(6) cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.

Improved functions of human hepatocytes on NH3 plasma-grafted PEEK-WC–PU membranes

PEEK-WC-PU membranes were modified with an NH(3) glow discharge process to graft N-containing functional groups at their surface in order to improve the maintenance of human hepatocytes. Native and modified membrane surfaces were characterized with XPS, ToF-SIMS and WCA measurements. We have investigated morphological behaviour and specific functions of primary human hepatocytes on native and modified PEEK-WC-PU membranes in a small-scale gas-permeable bioreactor. N-containing groups grafted at the surface of the membranes improved the initial steps of adhesion and the maintenance of phenotype and differentiated functions of cells. Confocal microscopy of cell morphology evidenced human hepatocytes exhibiting a polygonal shape and organizing a 3D structure. The presence of CK19 positive cells, a marker of biliary duct epithelium, was also found on native and modified membranes. Liver specific functions, investigated in terms of urea production, albumin synthesis and diazepam biotransformation, were maintained at high levels up to 19 days, particularly on surface-modified membranes.

Human hepatocytes and endothelial cells in organotypic membrane systems

The realization of organotypic liver model that exhibits stable phenotype is a major challenge in the field of liver tissue engineering. In this study we developed liver organotypic co-culture systems by using synthetic and biodegradable membranes with primary human hepatocytes and human umbilical vein endothelial cells (HUVEC). Synthetic membranes prepared by a polymeric blend constituted of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) and biodegradable chitosan membranes were developed by phase inversion technique and used in homotypic and organotypic culture systems. The morphological and functional characteristics of cells in the organotypic co-culture membrane systems were evaluated in comparison with homotypic cultures and traditional systems. Hepatocytes in the organotypic co-culture systems exhibit compact polyhedral cells with round nuclei and well demarcated cell-cell borders like in vivo, as a result of heterotypic interaction with HUVECs. In addition HUVECs formed tube-like structures directly through the interactions with the membranes and hepatocytes and indirectly through the secretion of ECM proteins which secretion improved in the organotypic co-culture membrane systems. The heterotypic cell-cell contacts have beneficial effect on the hepatocyte albumin production, urea synthesis and drug biotransformation. The developed organotypic co-culture membrane systems elicit liver specific functions in vitro and could be applied for the realization of engineered liver tissues to be used in tissue engineering, drug metabolism studies and bioartificial liver devices.

The influence of polymeric membrane surface free energy on cell metabolic functions

In membrane bioartificial organs using isolated cells, polymeric semipermeable membranes are used as immunoselective barriers, means for cell oxygenation and also as substrata for adhesion of anchorage-dependent cells. The selection of cytocompatible membranes that promote in vitro cell adhesion and function could be dependent on its membrane properties. In this study we investigated the physicochemical aspects of the interaction between the membrane and mammalian cells in order to provide guidelines to the selection of cytocompatible membranes. We evaluated the metabolic behavior of isolated liver cells cultured on various polymeric membranes such as the ones modified by protein adsorption. The physico-chemical properties of the membranes were characterized by contact angle measurements. The surface free energy of membranes and their different parameters acid (γ+), base (γ-) and Lifshitz-van der Waals (γLW) were calculated according to Good-van Osss model. The adsorption of protein modified markedly both contact angle and membrane surface tension. In particular, membrane surface free energy decreased drastically with increased water contact angle. For each investigated membrane we observed that liver specific functions of cells improve on hydrophilic membrane surfaces. For all investigated membranes the rate of ammonia elimination increased with increasing of membrane surface free energy.© 2001 Kluwer Academic Publishers

Influence of micro-patterned PLLA membranes on outgrowth and orientation of hippocampal neurites

In neuronal tissue engineering many efforts are focused on creating biomaterials with physical and chemical pathways for controlling cellular proliferation and orientation. Neurons have the ability to respond to topographical features in their microenvironment causing among others, axons to proliferate along surface features such as substrate grooves in micro-and nanoscales. As a consequence these neuronal elements are able to correctly adhere, migrate and orient within their new environment during growth. Here we explored the polarization and orientation of hippocampal neuronal cells on nonpatterned and micro-patterned biodegradable poly(l-lactic acid) (PLLA) membranes with highly selective permeable properties. Dense and porous nonpatterned and micro-patterned membranes were prepared from PLLA by Phase Separation Micromolding. The micro-patterned membranes have a three-dimensional structure consisting of channels and ridges and of bricks of different widths. Nonpatterned and patterned membranes were used for hippocampal neuronal cultures isolated from postnatal days 1-3 hamsters and the neurite length, orientation and specific functions of cells were investigated up to 12 days of culture. Neurite outgrowth, length plus orientation tightly overlapped the pattern of the membrane surface. Cell distribution occurred only in correspondence to membrane grooves characterized by continuous channels whereas on membranes with interconnected channels, cells not only adhered to and elongated their cellular processes in the grooves but also in the breaking points. High orientation degrees of cells were determined particularly on the patterned porous membranes with channel width of 20 mum and ridges of 17 mum whereas on dense nonpatterned membranes as well as on polystyrene culture dish (PSCD) controls, a larger number of primary developed neurites were distributed. Based on these results, PLLA patterned membranes may directly improve the guidance of neurite extension and thereby enhancing their orientation with a consequently highly ordered neuronal cell matrix, which may have strong bearings on the elucidation of regeneration mechanisms.

Improving the bioactivity of Zn(II)-curcumin based complexes

New Zn(II)-curcumin based heteroleptic complexes (1-5) have been synthesized and fully characterized, with the aim to improve the bioactivity of the precursor derivative [(bpy-9)Zn(curc)Cl] (A), a potentially intercalating antitumor agent recently reported. Some structural changes have been made starting from the reference complex A, in order to introduce new functionalities, such as electrostatic and/or covalent interactions. In particular, keeping the same N,N chelating ligand, namely bpy-9, two completely different Zn(II) species have been obtained: a tetracoordinated Zn(II) cation with tetrafluoroborate as counterion (1) and a dimeric neutral complex in which the sulfate anion acts as a bridging group through two Zn(II) centres (2). Moreover, by changing the N,N chelating unit, [(L(n))Zn(curc)Cl] complexes (3-5), in which the Zn(II) ion shows the same pentacoordination seen in the precursor complex A, have been obtained. The antitumour activity of all new Zn(II) complexes was tested in vitro against the human neuroblastoma cell line SH-SY5Y in a biohybrid membrane system and the results indicate that all species exhibit strong cytotoxic activity. In particular the ionic tetrafluoroborate Zn(II) complex, 1, and the neutral phenanthroline based Zn(II) derivative, 4, show the strongest growth inhibition, being even more effective than the model complex A. Both complexes have a dose-dependent anti-proliferative effect on cells as demonstrated by the decrease of viability and the increase of Annexin V and PI-positive cells with the increase of their concentration. Cells treated with complexes 1 and 4 undergo apoptosis that involves the activation of JNK, caspase 3 and MMP changes. Finally, complex 1 is more effective in the induction of caspase-3 activation demonstrating its ability to trigger the execution-phase of cell apoptosis.

Novel PEEK-WC membranes with low plasma protein affinity related to surface free energy parameters.

There has been growing interest in innovative materials with specific physico-chemical properties that provide an improved blood/cell compatibility. In this paper we evaluated the performance of new membranes prepared from a modified polyetheretherketone (PEEK-WC) contacting human plasma proteins. These membranes were prepared by using the phase inversion technique. Membrane wettability and affinity to proteins were evaluated by means of contact angle experiments, roughness measurements, and quantitative UV analysis. The energy parameters of membrane surfaces were determined according to Good, van Oss and Chaudhury’s theory. The extent of human albumin, fibrinogen and immunoglobulin G adsorption was related to quantitative expressions of the membrane surface hydrophilicity: the base parameter of surface free energy and the free energy of interfacial interaction. The performance of PEEK-WC membranes was compared to that of commercial membranes, which conventionally are used in biomedical applications. The experimental results showed a reduction of protein adsorption on PEEK-WC membranes with respect to other commercial membranes. The low protein affinity of PEEK-WC membranes is due to the intrinsic physico-chemical characteristics of the polymeric material which makes these membranes interesting for potential use in biomedical applications.

Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid–nanohydroxyapatite fiber scaffolds

The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation.

Biodegradable and synthetic membranes for the expansion and functional differentiation of rat embryonic liver cells

The insufficient availability of donor organs for orthotopic liver transplantation worldwide has urgently increased the requirement for new therapies for acute and chronic liver disease. The creation of an unlimited source of donor cells for hepatocyte transplantation therapy and pharmaceutical applications may be the isolation and expansion of liver progenitor cells or stem cells. Here we report the expansion and functional differentiation of rat embryonic liver cells on biodegradable and synthetic polymeric membranes in comparison with traditional substrates, such as collagen and polystyrene culture dishes. Membranes prepared from chitosan and modified polyetheretherketone were used for the culture of liver progenitor cells derived from rat embryonic liver. Cells proliferated, with a significant increase in their number within 8-11 days. The cells displayed functional differentiation showing urea synthesis, albumin production and diazepam biotransformation on all substrates investigated. In particular, on a chitosan membrane liver-specific functions were expressed at significantly higher levels for prolonged times compared with other synthetic membranes, utilizing traditional substrates (collagen and PSCD) as references. These results demonstrate that chitosan membranes offer cells favourable conditions to promote the expansion and functional differentiation of embryonic liver cells that could be effectively used in liver tissue engineering and in pharmaceutical applications.